Methods and compositions for neural disease immunotherapy

ABSTRACT

The invention provides antibodies to specific neural proteins and methods of using the same.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/456,642 filed on Nov. 10, 2010, U.S. Provisional Application No.61/418,310, filed Nov. 30, 2010, U.S. Provisional Application No.61/418,850, filed Dec. 1, 2010 and U.S. Provisional Application No.61/426,425, filed Dec. 22, 2010, all of which are incorporated byreference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to antibodies which are BACE1antagonists that, for example, inhibit or decrease BACE1 activity and tocompositions comprising such antibodies. Additional embodiments includemethods for treating and diagnosing various neurological diseases ordisorders, as well as methods of reducing APP and/or Aβ polypeptides ina patient.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

A sequence listing is submitted concurrently with the specification asan ASCII formatted text file via EFS-Web, with a file name of“P4453R1US.txt”, a creation date of Oct. 25, 2011, and a size of 116,192bytes. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated by reference in its entirety.

BACKGROUND

Amyloidosis is not a single disease entity but rather a diverse group ofprogressive disease processes characterized by extracellular tissuedeposits of a waxy, starch-like protein called amyloid, whichaccumulates in one or more organs or body systems. As the amyloiddeposits accumulate, they begin to interfere with the normal function ofthe organ or body system. There are at least 15 different types ofamyloidosis. The major forms are primary amyloidosis without knownantecedent, secondary amyloidosis following some other condition, andhereditary amyloidosis.

Many diseases of aging are based on or associated with amyloid-likeproteins and are characterized, in part, by the buildup of extracellulardeposits of amyloid or amyloid-like material that contribute to thepathogenesis, as well as the progression of the disease. These diseasesinclude, but are not limited to, neurological disorders such asAlzheimer's Disease (AD), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex. Other diseases which are based on orassociated with amyloid-like proteins are progressive supranuclearpalsy, multiple sclerosis, Creutzfeld Jacob disease, Parkinson'sdisease, HIV-related dementia, ALS (amyotropic lateral sclerosis), AdultOnset Diabetes, senile cardiac amyloidosis, endocrine tumors, andothers, including macular degeneration.

The polypeptide β-amyloid (Aβ) is likely to play a central role in thepathogenesis of Alzheimer's disease (AD). Vassar et al., J. Neurosci.29:12787-12794 (2009). Aβ polypeptide accumulation in the CNS results insynaptic dysfunction, axon degeneration and neuronal death. The brainsof AD patients show a characteristic pathology of prominentneuropathologic lesions, such as neurofibrillary tangles (NFTs), andamyloid-rich senile plaques. The major component of amyloid plaques isAβ. These lesions are associated with massive loss of populations ofcentral nervous system (CNS) neurons and their progression accompaniesthe clinical dementia associated with AD.

Aβ is the proteolytic product of the precursor protein, beta amyloidprecursor protein (β-APP or APP). APP is a type-I trans-membrane proteinwhich is sequentially cleaved by two proteases, a β- and γ-secretase.The β-secretase, known as β-site amyloid precursor protein cleavingenzyme 1 (BACE1), first cleaves APP to expose the N-terminus of Aβ,thereby producing a membrane bound fragment known as C99. Vassar et al.,J. Neurosci., 29:12787-12794 (2009) and UniProtKB/Swiss-Prot EntryP56817 (BACE1_HUMAN). The γ-secretase then is able to cleave C99 toproduce the mature Aβ polypeptide. Aβ is produced with heterogenous Ctermini ranging in length from 38 amino acids to 43 amino acids. The 42amino acid form of Aβ (Aβ₄₂) is the fibrillogenic form of Aβ and is overproduced in patients with Down's syndrome and has been suggested to playa role in the early pathogenesis of AD. Vassar et al., J. Neurosci.29:12787-12794 (2009). BACE1 has thus become a therapeutic target as itsinhibition would presumably inhibit APP and Aβ production.

Indeed, BACE1 knock-out mice (BACE1^(−/−)) do not produce cerebral Aβ,confirming that BACE1 is the major, if not only, enzyme responsible forproducing Aβ in the brain. Roberds et al., Human Mol. Genetics.10:1317-1324 (2001). Moreover, BACE1 knockout mice in AD models do notform amyloid plaques; cognitive defects and cholinergic dysfunction arerescued as well. McConlogue et al., J. Biol. Chem. 282: 26326-26334(2007); Ohno et al., Neuron 41: 27-33 (2004); and Laird et al., J.Neurosci. 25:11693-11709 (2005). Additionally, BACE1 heterozygousknock-out mice have reduced plaque formation indicating the completeinhibition of BACE1 activity is not necessary for plaque reduction.McConlogue et al., J. Biol. Chem. 282: 26326-26334 (2007).

Recently, APP has been shown to be a ligand for Death Receptor 6 (DR6)which triggers caspase-dependent neuronal cell body death and axonpruning Nikolaev et al., Nature 457: 981-989 (2009). In addition, aBACE1 compound inhibitor impaired degeneration of axons and cell bodies.Id. These results point to a model in which APP, via DR6 binding maycontribute to AD.

It would be beneficial to have an effective therapeutic inhibitor ofBACE1 to reduce APP and Aβ production in patients with neurologicaldiseases and disorders, such as AD. The invention provided hereinrelates to such inhibitors, including their use in a variety of methods.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

SUMMARY

The invention provides BACE1 antagonist antibodies and methods of usingthe same. Specifically, the antibodies inhibit or reduce the activity ofBACE1.

In one embodiment, an isolated antibody that binds to BACE1, wherein theantibody reduces or inhibits the activity of the BACE1 polypeptide isprovided. In particular, the antibody binds to the active site of BACE1or to an exosite of BACE1.

In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises at least one hypervariable region (HVR)sequence selected from the group consisting of SEQ ID NOs: 7-19, 22-26,28-30, 35-47, 56-79 and 118-122.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises at least one sequence selected from thegroup consisting of HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprisesthe amino acid sequence GFX₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO:45), whereinX₃₀=N or T; X₃₁=S, L or Y; X₃₂=G or Y; X₃₃=Y or S; and X₃₄=A, G or S;HVR-H2 comprises the amino acid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG(SEQ ID NO:46), wherein X₃₅=A or G; X₃₆=W or 5; X₃₇=A or Y; X₃₈=G or 5;X₃₉=S or Y; and X₄₀=D or 5; and HVR-H3 comprises the amino acid sequenceX₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO:47), wherein X₄₁=Q or G; X₄₂=T orF; X₄₃=H or 5; X₄₄=Y or P; X₄₅=Y or W; X₄₆=Y or V and wherein X₄₇optionally includes the sequence YAKGYKA (SEQ ID NO:48). Alternatively,the antibody comprises an HVR-H1 sequence comprising the amino acidsequence GFTFX₁₃GYX₁₄IH (SEQ ID NO:26), wherein X₁₃=S or L and X₁₄=A orG; or an amino acid sequence selected from the group consisting of SEQID NO:22; SEQ ID NO:23; and SEQ ID NO:28.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises at least one sequence selected from thegroup consisting of HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprisesthe amino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO:120),wherein X₇₁=F or Y; X₇₂=F, N or T; X₇₃=F or Y; X₇₄=1, Q, I, S or Y;X₇₅=G or Y; X₇₆=Y or S; and X₇₇=A, G or S; HVR-H2 comprises the aminoacid sequence X₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO:121), whereinX₇₈=A or G; X₇₉=W or S; X₈₀=A, S, Q or Y; X₈₁=G or S; X₈₂=S, K, L or Y;X₈₃=T or Y; and X₈₄=D or S; and HVR-H3 comprises the amino acid sequenceX₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO:122), wherein X₈₅=Q or G; X₈₆=T orF; X₈₇=H, Y or S; X₈₈=Y or P; X₈₉=Y or W; X₉₀=Y or V and wherein X₉₁optionally includes the sequence YAKGYKA (SEQ ID NO:48). Alternatively,the antibody comprises an HVR-H1 sequence comprising the amino acidsequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO:68), wherein X₅₃=F or Y; X₅₄=T orF; X₅₅=F or Y; X₅₆=L, Q or I; or an amino acid sequence selected fromthe group consisting of SEQ ID NOs:71-73. Alternatively, the antibodycomprises an HVR-H2 sequence comprising the amino acid sequenceGWISPX₅₇X₅₈GX₅₉X₆₀DYADSVKG (SEQ ID NO:69), wherein X₅₇=A, S or Q; X₅₈=Gor S; X₅₉=S, K or L; X₆₀=T or Y; or an amino acid sequence selected fromthe group consisting of SEQ ID NOs:74-78. Alternatively, the antibodycomprises an HVR-H3 sequence comprising the amino acid sequenceGPFX₆₁PWVMDY (SEQ ID NO:70), wherein X₆₁=S or Y; or an amino acidsequence of SEQ ID NO:79.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-H1 sequence comprising an amino acidsequence selected from SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 and SEQID NOs:71-73.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-H2 sequence comprising an amino acidsequence selected from SEQ ID NO:24, SEQ ID NO:29 and SEQ ID NOs:74-78.

In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-H3 sequence comprising an aminoacid sequence selected from SEQ ID NO:25, SEQ ID NO:30 and SEQ ID NO:79.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises HVR-H1, HVR-H2, and HVR-H3 sequencescorresponding to those set forth for clones YW412.8, YW412.8.31,YW412.8.30, YW412.8.2, YW412.8.29 and YW412.8.51 in FIG. 1(B) or thoseset forth for clones Fab12, LC6, LC9 and LC10 in FIG. 2(B) or thoseclones set forth in FIGS. 24A-C.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-H1 sequence of SEQ ID NO:22 or 23, anHVR-H2 sequence of SEQ ID NO:24 and an HVR-H3 sequence of SEQ ID NO:25.In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-H1 sequence of SEQ ID NO:23, anHVR-H2 sequence of SEQ ID NO:24 and an HVR-H3 sequence of SEQ ID NO:25.In yet another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-H1 sequence of SEQ ID NO:28, anHVR-H2 sequence of SEQ ID NO:29, and an HVR-H3 sequence of SEQ ID NO:30.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-H1 sequence selected from SEQ IDNOs:71-73, an HVR-H2 sequence selected from SEQ ID NOs:74-78 and anHVR-H3 sequence selected from SEQ ID NO:79.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises a variable heavy (VH) chain having an aminoacid sequence selected from the group consisting of SEQ ID NOs: 20, 21,27 and 80-98. In one aspect, the antibody comprises the VH chain aminoacid sequence of SEQ ID NO:21.

In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises at least one sequence selected from thegroup of HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:42), wherein X₁₇=S, D orV; X₁₈=S or A; X₁₉=S, T or N; X₂₀=A or S; X₂₁=V or L, HVR-L2 comprisesthe amino acid sequence X₂₂ASX₂₃LYS (SEQ ID NO:43), wherein X₂₂=S, W, Yor L; X₂₃=F, S or W, and HVR-L3 comprises the amino acid sequenceQQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T (SEQ ID NO:44), wherein X₂₄=S, F, G, D or Y;X₂₅=Y, P, S or A; X₂₆=Y, T or N; X₂₇=T, Y, D or S; X₂₈=P or L; andX₂₉=F, P or T.

In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises at least one sequence selected from thegroup of HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:42), wherein X₁₇=S, D orV; X₁₈=S or A; X₁₉=S, T or N; X₂₀=A or S; X₂₁=V or L, HVR-L2 comprisesthe amino acid sequence X₆₂ASX₆₃X₆₄YX₆₅ (SEQ ID NO:118), wherein X₆₂=S,W, Y, F or L; X₆₃=F, S, Y or W; X₆₄=L or R; X₆₅=S, P, R, K or W, andHVR-L3 comprises the amino acid sequence QQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ IDNO:119), wherein X₆₆=S, F, G, D or Y; X₆₇=Y, P, S or A; X₆₈=Y, T or N;X₆₉=T, Y, D or S; X₇₀=P, Q, S, K or L; and X₇₁=F, P or T.

In certain embodiments, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-L1 sequence comprising the aminoacid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V; X₂=5or A; X₃=T or N; X₄=5 or A; X₅=V or L or an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:35.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-L2 sequence comprising the amino acidsequence X₆ASFLYS (SEQ ID NO:18), wherein X₆=S or L or X₁₅ASX₁₆LYS (SEQID NO:41), wherein X₁₅=S, W or Y and X₁₆=S or W or an amino acidsequence selected from the group consisting of SEQ ID NO:9, SEQ IDNO:10, and SEQ ID NOs:36-39.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-L3 sequence comprising the amino acidsequence QQX₇X₈X₉X₁₀X₁₁X₁₂T (SEQ ID NO:19), wherein X₇=S, F, G, D or Y;X₈=Y, P, S, or A; X₉=T or N; X₁₀=T, Y, D or S; X₁₁=P or L; X₁₂=P or T oran amino acid sequence selected from the group consisting of SEQ IDNOs:11-16 and SEQ ID NO:40.

In certain embodiments, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-L1 sequence comprising the aminoacid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V; X₂=5or A; X₃=T or N; X₄=5 or A; X₅=V or L or an amino acid sequence selectedfrom the group consisting of SEQ ID NO:7.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-L2 sequence comprising the amino acidsequence X₄₈ASX₄₉X₅₀YX₅₁ (SEQ ID NO:56), wherein X₄₈=S or F; X₄₉=F or Y;X₅₀=L or R; X₅₁=S, P, R, K or W or an amino acid sequence selected fromthe group consisting of SEQ ID NOs:58-64.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-L3 sequence comprising the amino acidsequence QQFPTYX₅₂PT (SEQ ID NO:57), wherein X₅₂=L, Q, S or K or anamino acid sequence selected from the group consisting of SEQ IDNOs:65-67.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises HVR-L1, HVR-L2 and HVR-L3 sequencescorresponding to those set forth for clones YW412.8, YW412.8.31,YW412.8.30, YW412.8.2, YW412.8.29 and YW412.8.51 in FIG. 1(A) or thoseset forth for clones Fab12, LC6, LC9 and LC10 in FIG. 2(A) or those setforth for clones in FIG. 23A-C.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises an HVR-L1 sequence of SEQ ID NO:7 or SEQ IDNO:8; an HVR-L2 sequence selected from the group consisting of SEQ IDNO:9, SEQ ID NO:10 or SEQ ID NOs: 58-64; and an HVR-L3 sequence selectedfrom the group consisting of: SEQ ID NOs:11-16 and 65-67. In anotheraspect, an isolated antibody that binds to BACE1 is provided wherein theantibody reduces or inhibits the activity of the BACE1 polypeptide andcomprises an HVR-L1 sequence of SEQ ID NO:7, an HVR-L2 sequence of SEQID NO:9 and an HVR-L3 sequence of SEQ ID NO:12.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-L1 sequence of SEQ ID NO:7, SEQID NO:8 or SEQ ID NO:35.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-L2 sequence of SEQ ID NOs:9-10,36-39 or 58-64.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-L3 sequence of SEQ ID NOs: 11-16,40 or 65-67.

In another embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises a variable light (VL) chain sequencehaving an amino acid sequence selected from the group consisting of: SEQID NOs: 1-6, 31-34 and 99-117. In one aspect, the VL chain amino acidsequence is SEQ ID NO:2.

In an additional embodiment, an isolated antibody that binds to BACE1 isprovided wherein the antibody reduces or inhibits the activity of theBACE1 polypeptide and comprises an HVR-H1 sequence of SEQ ID NO:23, anHVR-H2 sequence of SEQ ID NO:24, an HVR-H3 sequence of SEQ ID NO:25, anHVR-L1 of SEQ ID NO:7, an HVR-L2 of SEQ ID NO:9 and an HVR-L3 of SEQ IDNO:12.

In one embodiment, an isolated antibody that binds to BACE1 is providedwherein the antibody reduces or inhibits the activity of the BACE1polypeptide and comprises a VL chain comprising the amino acid sequenceof SEQ ID NO:2 and a VH chain comprising the amino acid sequence of SEQID NO:21.

In another embodiment, an isolated antibody which binds to an epitopecomprising at least one of the amino acid residues of BACE1 selectedfrom the group consisting of: 314 SER; 316 GLU; 317 LYS; 327 GLN; 330CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49 is provided.In certain embodiments, the antibody binds to an epitope of BACE1comprising the amino acids: 314 SER; 316 GLU; 317 LYS; 327 GLN; 330 CYS;331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49.

In other embodiments, the antibody binds to an epitope of BACE1comprising at least one amino acid region of BACE1 selected from thegroup consisting of: amino acids 315-318 of SEQ ID NO:49; amino acids331-335 of SEQ ID NO:49; amino acids 370-381 of SEQ ID NO:49; and anycombination thereof. In one embodiment, the antibody binds to an epitopeof BACE1 comprising amino acids 315-318, 331-335 and 370-381 of SEQ IDNO:49.

In another embodiment the antibody binds to an epitope of BACE1 whichresults in a conformational change in the structure of the P6 and P7sites of BACE1 upon binding. In an additional embodiment, the antibodybinds to an epitope of BACE1 which induces amino acids 218-231 of SEQ IDNO:49 to adopt a random loop structure.

An antibody of the invention can be in any number of forms. For example,an antibody of the invention can be a human antibody, humanized antibodyor chimeric antibody. In other aspects the antibody of the invention isa full length antibody or a fragment thereof (e.g., a fragmentcomprising an antigen binding component). In other aspects of theinvention, the antibody is a monoclonal antibody. In another aspect, anantibody of the invention can be linked or conjugated to an agent ormoiety, e.g. a cytotoxic agent, to create an immunoconjugate.

In one embodiment, a pharmaceutical formulation is provided whichcomprises an antibody of the invention and a pharmaceutically acceptablecarrier. In additional embodiments an isolated nucleic acid encoding anantibody of the invention is provided, as well as vector that comprisesthe nucleic acid encoding an antibody of the invention. In anotheraspect, a host cell comprising the nucleic acid encoding an antibody ofthe invention is provided as well as methods for producing an antibodyof the invention comprising culturing the host cell comprising thenucleic acid encoding an antibody of the invention under conditionssuitable for production of the antibody.

In another embodiment, a method of treating an individual having aneurological disease or disorder comprising administering to theindividual an effective amount of an antibody of the invention isprovided.

In an additional embodiment, a method of reducing amyloid plaques, orinhibiting amyloid plaque formation, in a patient suffering from, or atrisk of contracting, a neurological disease or disorder comprisingadministering to the individual an effective amount of an antibody ofthe invention is provided.

In one embodiment, a method of reducing Aβ protein in a patientcomprising administering to the patient an effective amount of anantibody of the invention. In one aspect, the patient is suffering from,or at risk of contracting, a neurological disease or disorder.

In another embodiment, a method of inhibiting axon degeneration in apatient comprising administering to the patient an effective amount ofan antibody of the invention is provided.

In an additional embodiment, a method of diagnosing a neurologicaldisease or disorder in patient comprising contacting a biological sampleisolated from the patient with an antibody of the invention underconditions suitable for binding of the antibody to a BACE1 polypeptide,and detecting whether a complex is formed between the antibody and theBACE1 polypeptide.

In one embodiment, a method of determining whether a patient is eligiblefor therapy with an anti-BACE1 antibody, comprising contacting abiological sample isolated from the patient with an antibody of theinvention under conditions suitable for binding of the antibody to aBACE1 polypeptide, and detecting whether a complex is formed between theantibody and the BACE1 polypeptide, wherein the presence of a complexbetween the antibody and BACE1 is indicative of a patient eligible fortherapy with an anti-BACE1 antibody. In one aspect the patient issuffering from, or at risk of contracting, a neurological disease ordisorder.

In one aspect, biological samples that may be used in the diagnosis of aneurological disease or condition; or for predicting responsiveness, ordetermining eligibility, of a patient to a treatment with a BACE1antibody include, but are not limited to, fluids such as serum, plasma,saliva, gastric secretions, mucus, cerebrospinal fluid, lymphatic fluidand the like or tissue or cell samples obtained from an organism such asneuronal, brain, cardiac or vascular tissue.

In one aspect of the methods of the invention, the patient is mammalian.In another aspect, the patient is human. In another aspect, theneurological disease or disorder is selected from the group consistingof Alzheimer's disease (AD), traumatic brain injury, stroke, glaucoma,dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophiclateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddlesyndrome, Paget's disease, traumatic brain injury, Lewy body disease,postpoliomyelitis syndrome, Shy-Draeger syndrome, olivopontocerebellaratrophy, Parkinson's disease, multiple system atrophy, striatonigraldegeneration, supranuclear palsy, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, fatalfamilial insomnia, bulbar palsy, motor neuron disease, Canavan disease,Huntington's disease, neuronal ceroid-lipofuscinosis, Alexander'sdisease, Tourette's syndrome, Menkes kinky hair syndrome, Cockaynesyndrome, Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome, dementia (including, but not limited to,Pick's disease, and spinocerebellar ataxia). In one aspect, theneurological disease or disorder is Alzheimer's disease.

In one embodiment, a BACE1 epitope which is specifically recognized byan antibody, or fragment thereof, comprising at least one of the aminoacid residues of BACE1 which correspond to the amino acids selected fromthe group consisting of: 314 SER; 316 GLU; 317 LYS; 327 GLN; 330 CYS;331 TRY; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49 is provided. Inone aspect, the BACE1 epitope comprises amino acids which correspond to314 SER; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRY; 332 GLN; 335 THR;and 378 ASP of SEQ ID NO:49.

In one embodiment, a BACE1 epitope which is specifically recognized byan antibody, or fragment thereof, comprising at least one amino acidregion of BACE1 selected from the group consisting of: amino acids315-318 of SEQ ID NO:49; amino acids 331-335 of SEQ ID NO:49; aminoacids 370-381 of SEQ ID NO:49; and any combination thereof. In oneaspect, the BACE1 epitope comprises amino acids 315-318, 331-335 and370-381 of SEQ ID NO:49.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B depict the light and heavy chain amino acid sequences ofclone YW412.8 obtained from a naïve sort of the natural diversity phagedisplay library and affinity-matured forms of YW412.8 as described inExample 1(A). FIG. 1A depicts the light chain sequence alignments. FIG.1B depicts the heavy chain sequence alignments. In both FIGS. 1A and 1B,the HVR sequences for each clone are indicated by the boxed regions,with the first box indicating HVR-L1 (SEQ ID NOs:7 and 8—FIG. 1A) orHVR-H1 (SEQ ID NOs:22 and 23-FIG. 1B), the second box indicating HVR-L2(SEQ ID NOs:9 and 10—FIG. 1A) or HVR-H2 (SEQ ID NO:24—FIG. 1B), and thethird box indicating HVR-L3 (SEQ ID NOs:11-16—FIG. 1A) or HVR-H3 (SEQ IDNO:25—FIG. 1B).

FIGS. 2A-2B depict the light and heavy chain amino acid sequences ofclone Fab 12 obtained from a naïve sort of a synthetic diversity phagedisplay library and affinity-matured forms of Fab 12, as described inExample 1(B). FIG. 2A depicts the light chain sequence alignments. FIG.2B depicts the heavy chain sequence alignments. In both FIGS. 2A and 2B,the HVR sequences for each clone are indicated by the boxed regions,with the first box indicating HVR-L1 (SEQ ID NO:35—FIG. 2A) or HVR-H1(SEQ ID NO:28—FIG. 2B), the second box indicating HVR-L2 (SEQ IDNOs:36-39—FIG. 2A) or HVR-H2 (SEQ ID NO:29-FIG. 2B), and the third boxindicating HVR-L3 (SEQ ID NO:40—FIG. 2A) or HVR-H3 (SEQ ID NO:30—FIG.2B).

FIGS. 3A and 3B depict the HVR or CDR sequences from the light and heavychain Fabs isolated from the synthetic diversity phage display libraryas described in Example 1(B). The numbering is according to thenomenclature of Kabat et al. FIG. 3A discloses the “CDRL1” sequences asSEQ ID NO: 133, the “CDRL2” sequences as SEQ ID NO: 134, the “CDRL3”sequences as SEQ ID NOS 135-144, 141, and 145-152 and the “CDRH1”sequences as SEQ ID NOS 153-159, 158, 160-161, 159, 158, 162, 161, and163-167 all, respectively, in order of appearance. FIG. 3B discloses the“CDRH2” sequences as SEQ ID NOS 168-177, 174, 171, 178-182, 177, and183, and the “CDRH3” sequences as SEQ ID NOS 184-202, all, respectively,in order of appearance.

FIG. 4 provides a graph showing the inhibition of BACE1 by the variousclones identified from the natural diversity and synthetic diversityphage display libraries. The clones were tested for BACE1 inhibition ina homogenous time-resolved fluorescence (HTRF) assay, as described inExample 1(A). All YW series antibodies were used at a concentration of500 nM except for the YW 434.6 antibody, which was tested at aconcentration of 320 nM. Antibodies 12.IgG, 14.IgG LC6.IgG, LC9.IgG,LC10.IgG and LC11.IgG were tested at 1 μM concentration.

FIG. 5 is a graph showing the activity of BACE1 in an HTRF assay in thepresence of anti-BACE1 Fabs identified from the synthetic diversityphage display library, as described in Example 1(B). Lines correspond to100% activity (0% inhibition) in the presence of BACE1 and substrate(PBS Control) and 100% inhibition in the absence of BACE1.

FIG. 6 depicts the CDR or HVR sequences of affinity matured anti-BACE1Fabs as described in Example 1(B). The numbering is according to thenomenclature of Kabat et al. The competition ELISA ratio is the ratio ofELISA signal in the absence or presence of 20 nM BACE1 as competitor insolution in one-point competition ELISA assays as described in Example1(B). FIG. 6 discloses the “CDRL1” sequences as SEQ ID NOS 133, 133,133, 133, 133, and 203, the “CDRL2” sequences as SEQ ID NOS 134, 134,and 204-207, the “CDRL3” sequences as SEQ ID NOS 208-209, 145, 145, and145-146, the “CDRH1” sequences as SEQ ID NOS 157, 157, 158, 158, 158,and 162, the “CDRH2” sequences as SEQ ID NOS 172, 172, 171, 171, 171,and 178, and the “CDRH3” sequences as SEQ ID NOS 188, 188, 195, 195, and195-196, all, respectively, in order of appearance.

FIGS. 7A-7C contain graphs displaying data from competitive ELISA assayswith affinity matured anti-BACE clones as described in Example 1(B). Thebinding between Fab-displaying phage and BACE1-immobilized on plates wascompeted with serial dilution of BACE1 in solution. FIGS. 7A, 7B and 7Cdepict competition curves for the parent and corresponding affinitymatured antibodies.

FIGS. 8A-8C depict graphs showing the inhibition of BACE1 withanti-BACE1 Fabs in an HTRF enzyme assay as described in Example 1(B).The inhibition activity of purified Fabs for individual anti-BACE1clones were measured in an HTRF enzyme assay. OM99-2 (CalBiochem®,catalog #496000), is a synthetic peptide inhibitor for BACE1 and wasused as a positive control. FIGS. 8A, 8B and 8C are inhibition curvesfor the parent and corresponding affinity matured derivatives. The IC₅₀for OM99-2 was 11 nM in this assay.

FIG. 9A provides a graph showing the impact of the affinity maturedYW412.8.31 anti-BACE1 antibody on the in vitro enzymatic activity ofhuman recombinant BACE1 using either a long peptide substrate withenhanced susceptibility to BACE1 in an HTRF assay (left panel) or ashort peptide substrate with enhanced susceptibility to BACE1 in a FRETassay (right panel), as described in Example 2(B). OM99-2 (CalBiochem®,catalog #496000), a synthetic peptide inhibitor of BACE1, β-Secretaseinhibitor IV (CalBiochem®, catalog #565788), a small molecule inhibitorof BACE1 (BACE1 SMI) and an IgG antibody which does not bind BACE1 wereused as controls. FIGS. 9B-1 and 9B-2 also provide graphs showing the invitro enzymatic activity of human recombinant BACE1 extracellulardomain, human recombinant BACE2 extracellular domain, or the cathepsin Dextracellular domain on a short peptide substrate with enhancedsusceptibility to BACE1 in the presence of YW412.8.31, or a control IgGantibody as described in Example 2(B).

FIG. 10 depicts the results of experiments performed with variousanti-BACE1 antibodies (LC6, LC9, YW412.8, YW412.8.30, YW412.8.31 andYW412.8.51) on the processing of recombinant amyloid precursor protein(APP) in 293-HEK cells, as described in Example 2(C). An IgG antibodywhich does not bind BACE1 (Xolair®) was used as a control.

FIGS. 11A-11D provide graphs illustrating the effects of the YW412.8.31anti-BACE1 antibody on processing of recombinant or endogenous amyloidprecursor protein (APP), as described in Example 2(C). FIG. 11A showsresults from experiments using 293-HEK cells stably expressing wild-typehuman APP. BACE1 SMI is a small molecule BACE1 inhibitor which was usedas a control (Compound 8e—Charrier et al., J. Med. Chem. 51:3313-3317(2008). FIG. 11B shows results from experiments using E13.5 dorsal rootganglia neurons cultured from wild-type CD1 mice. Additional experimentswere performed using cultures of E16.5 cortical neurons (FIG. 11C) andE16.5 cultured hippocampal neurons (FIG. 11D) from wild-type CD1 mice.

FIGS. 12A-12C provide images of YW412.8.31 anti-BACE1 antibody uptakeinto primary mouse neurons, as described in Example 2(D). FIG. 12A showsinternalization of YW412.8.31 anti-BACE1 antibody into intracellularvesicles in neurons. Embryonic cortical neurons were incubated at 37° C.for the times indicated. Bound YW412.8.31 was detected on surface(non-permeabilized) or internal (permeabilized) cellular compartmentswith α-human-Alexa 568. The majority of signal was internalized.Internalized YW412.8.31 was localized to subcellular compartments byco-staining with the indicated markers for vascular compartments: earlyendosomes (TfR); trans-golgi network (VAMP4) and lysosome (LAMP1). Scalebar=65 μm (top) and 20 μm (bottom). FIG. 12B shows uptake of anti-BACE1antibody into E13.5 dorsal root ganglion (DRG) neurons at two differenttemperatures and three different time points, as indicated in thefigure. Cells were permeabilized to allow for labeling of intracellularBACE1 antibody. Only externally bound YW412.8.31 anti-BACE1 antibody islabeled in the non-permeabilized cells. FIG. 12(C) shows uptake intoE16.5 cortical neurons of YW412.8.31 anti-BACE1 antibody fromBACE1-expressing or BACE1 knockout mice.

FIG. 13 provides a graphical representation of ELISA results fromExample 3(A), comparing the competitive binding of YW412.8 anti-BACE1antibody, with itself, another anti-BACE1 antibody (LC6), an active-siteBACE1 binding peptide (OM99-2 (CalBiochem®, catalog #496000)) and anexosite BACE1 binding peptide (BMS1) (Peptide 1 in Kornacker et al.,Biochemistry 44:11567-11572 (2005)).

FIG. 14 shows different views of the 2.8 Å structure of the FabYW412.8.31 co-crystallized with the human BACE1 extracellular domain asdescribed in Example 3(B). The Fab binds to a BACE1 exosite distal tothe secretase active site, partially overlapping with another exositeknown to interact with certain peptides having BACE1-inhibitoryproperties.

FIG. 15 provides a close-up view of the interaction of the FabYW412.8.31 with the human BACE1 extracellular domain. BACE1 is shown insurface representation and the Fab is shown as ribbons. The dottedsurface indicates the BACE1 epitope.

FIGS. 16A and 16B show the results of experiments to examine thecontribution of BACE1 to Aβ₁₋₄₀ levels in wild-type mice. Aβ₁₋₄₀ levelsin BACE1+/+ vs. BACE1−/− mice were examined. Mice were dosed with asingle dose of control IgG antibody or anti-BACE YW412.8.31 antibody asdescribed in Example 4. FIG. 16A shows the results of genetic studiesexamining the contribution of BACE1 to Aβ₁₋₄₀ production in mice. Levelsof Aβ₁₋₄₀ observed in BACE1 knockout mice (BACE1−/−) provide a controlfor how specific inhibitors of BACE1 alter Aβ₁₋₄₀ production inwild-type mice. FIG. 16B shows effects of dosing control IgG oranti-BACE1 YW412.8.31 (50 mg/kg) on Aβ₁₋₄₀ production in plasma and CNS(cortex) 24 or 48 hours after dosing. A single dose of control IgG oranti-BACE1 antibody (50 mg/kg) was delivered by IV injection to C57Bl/6mice. 24 or 48 hours later, plasma and brain samples were harvested toanalyze Aβ₁₋₄₀. Plasma Aβ₁₋₄₀ is reduced by 35% (at 24 hr) and corticalAβ₁₋₄₀ by ˜20%. Values plotted are mean (±SEM)* p<0.01; ** p<0.001.

FIGS. 17A-17B provide results of the in vivo YW412.8.31 anti-BACE1antibody experiments described in Example 4. FIG. 17A shows plots ofAβ₁₋₄₀ levels observed in the plasma and the hippocampus of mice treatedwith the YW412.8.31 anto-BACE1 antibody at two different concentrationsin comparison to vehicle control treatment. FIG. 17B is a plot ofindividual pharmacokinetic versus pharmacodynamic readouts, indicatingthat a PK/PD relationship exists in this mouse model for the YW412.8.31anti-BACE1 antibody.

FIGS. 18A and 18B show a comparison from experiments in whichhAPP-transgenic mice were dosed with the YW412.8.31 anti-BACE1 antibodysystemically (Panel A, same experiment as described in FIG. 17Aregraphed for comparison) or by continuous ICV infusion (Panel B). InFIG. 18A, Animals received vehicle or anti-BACE1 antibody (30 or 100mg/kg) by IP injection (3 doses @ Q4D). 2 hours after the last dose,plasma and brain samples were harvested to analyze Aβ₁₋₄₀ and Aβ₁₋₄₂.Plasma Aβ₁₋₄₀ and Aβ₁₋₄₂ were reduced to ˜30% control levels at both 30and 100 mg/kg anti-BACE1 antibody. Hippocampal Aβ₁₋₄₀ and Aβ₁₋₄₂ werereduced (13-22%) by the high dose of anti-BACE1 (100 mg/kg), andcortical Aβ₁₋₄₀ and Aβ₁₋₄₂ showed a trend toward reduction (12-18%). InFIG. 18B, Control IgG or anti-BACE1 antibody was delivered by unilateralICV infusion for 7 days. Consistent reductions were seen in Aβ₁₋₄₀ andAβ₁₋₄₂ at both doses in cortex (15-23%) and in hippocampus (15-20%).Panel C shows the levels of anti-BACE1 antibody in the brain followingsystemic vs. ICV delivery. Values plotted are mean (±SEM)* p<0.05; **p<0.001

FIGS. 19A and 19B show the PK analysis of a single dose of YW412.8.31anti-BACE1 (1 or 10 mg/kg) delivered via IV injection to BALB/C mice(FIG. 19A). Serum PK was analyzed out to 21 days post-dose. Two separatePK assays were used: an assay to detect all anti-BACE1 in serum (totalmAb), and an assay to detect only unbound anti-BACE1 in serum (freemAb). Single dose PK analysis in BACE1+/+, BACE1+/−, and BACE1−/− miceconfirms the non-linearity observed in the initial study, and indicatesthat the enhanced clearance is indeed target-mediated (FIG. 19B).

FIGS. 20A and 20B show the PK analysis of Cynomolgus monkeys dosed withcontrol IgG or YW412.8.31 anti-BACE1 antibody (30 mg/kg) by IV delivery.Total anti-BACE1 or control antibody concentrations in monkey serum(FIG. 20A) and CSF samples (FIG. 20B) were measured usingmonkey-adsorbed goat anti-human IgG polyclonal antibody (Bethyl,Montgomery, Tex.) as described in Example 5.

FIGS. 21A-21D are the results of experiments as described in Example 5in which Cynomolgus monkeys were dosed with control IgG or anti-BACE1antibody YW412.8.31 by IV delivery. Hatched lines show data forindividual animals, and solid lines show group means. Plasma and CSFwere sampled 7 days, 2 days and just prior to dosing to set a mean valuefor Aβ₁₋₄₀ baseline levels in each individual monkey. Plasma Aβ₁₋₄₀(FIG. 21A) and CSF Aβ₁₋₄₀ (FIG. 21B) was measured at various times. Thevariability across animals in baseline plasma (FIG. 21C) and CSF (FIG.21D) Aβ₁₋₄₀ is also shown.

FIGS. 22A and 22B depict Aβ production following systemic dosing ofYW412.8.31 in wild-type mice. FIG. 22A is a graph showing Aβ₁₋₄₀production following a single dose of control IgG or YW412.8.31 (100mg/kg) administered by IP injection to C57Bl/6J mice. 4 hours later,plasma and brain samples were harvested to analyze Aβ₁₋₄₀. Plasma Aβ₁₋₄₀is reduced by 48%, but forebrain Aβ₁₋₄₀ is not reduced in this paradigm.FIG. 22B is a graph showing Aβ₁₋₄₀ production following control IgG orYW412.8.31 (30 or 100 mg/kg) administration by 3 IP injections, each 4days apart. 4 hours after the last dose, plasma and brain samples wereharvested to analyze Aβ₁₋₄₀. Plasma Aβ₁₋₄₀ is reduced by 50-53%, whereasforebrain Aβ₁₋₄₀ is not reduced by dosing at 30 mg/kg, but is reduced by42% when dosed at 100 mg/kg. Values plotted are mean (±SEM)* p<0.0001

FIGS. 23A-23C depict the light chain amino acid sequences of cloneYW412.8.31 and affinity-matured forms of YW412.8.31. FIGS. 23A-23Cdepict the complete light chain sequence alignments. The HVR sequencesfor each clone are indicated by the boxed regions, with the first boxindicating HVR-L1 (SEQ ID NO:7—FIG. 23A), the second box indicatingHVR-L2 (SEQ ID NOs:9 and 58-64—FIG. 23B), and the third box indicatingHVR-L3 (SEQ ID NOs:12 and 66-67—FIG. 23C).

FIGS. 24A-24C depict the heavy chain amino acid sequences of cloneYW412.8.31 and affinity-matured forms of YW412.8.31. FIGS. 24A-24Cdepict the complete heavy chain sequence alignments. The HVR sequencesfor each clone are indicated by the boxed regions, with the first boxindicating HVR-H1 (SEQ ID NOs:24 and 71-73—FIG. 24A), the second boxindicating HVR-H2 (SEQ ID NOs:24 and 74-78—FIG. 24B), and the third boxindicating HVR-H3 (SEQ ID NOs:25 and 79—FIG. 24C).

FIGS. 25A and B depict graphs showing the inhibition of BACE1 withYW412.8.31 and affinity matured clones in an HTRF assay as described inExample 6. The ability of clones YW412.8.31.35; YW412.8.31.95;YW412.8.31.255; YW412.8.31.585; YW412.8.31.53; YW412.8.31.69;YW412.8.31.77; YW412.8.31.81S and YW412.8.31.895 to inhibit the proteaseactivity of BACE1 was tested.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The terms “anti-beta-secretase antibody”, “anti-BACE1 antibody”, “anantibody that binds to beta-secretase” and “an antibody that binds toBACE1” refer to an antibody that is capable of binding BACE1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting BACE1. In one embodiment, theextent of binding of an anti-BACE1 antibody to an unrelated, non-BACE1protein is less than about 10% of the binding of the antibody to BACE1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to BACE1 has a dissociation constant (Kd) of ≦1μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸Mor less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). Incertain embodiments, an anti-BACE1 antibody binds to an epitope of BACE1that is conserved among BACE1 from different species and isoforms.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited toradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-BACE1 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “BACE1,” as used herein, refers to any native beta-secretase 1(also called β-site amyloid precursor protein cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2or Asp2) from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed BACE1 as wellas any form of BACE1 that results from processing in the cell. The termalso encompasses naturally occurring variants of BACE1, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryBACE1 polypeptide is shown in SEQ ID NO:49 below, and is the sequencefor human BACE1, isoform A as reported in Vassar et al., Science286:735-741 (1999), which is incorporated herein by reference in itsentirety.

(SEQ ID NO: 49) MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLRLPRETDEEPEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFAVGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLVSIPHGPNVTVRANIAAITESDKFFINGSNWEGILGLAYAEIARPDDSLEPFFDSLVKQTHVPNLFSLQLCGAGFPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEINGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFPDGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRPVEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSACHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFMLPLCLMVCQWCCLRCLRQQHDDFADDISLL K

Several other isoforms of human BACE1 exist including isoforms B, C andD. See UniProtKB/Swiss-Prot Entry P56817, which is incorporated hereinby reference in its entirety. Isoform B is shown in SEQ ID NO:50 anddiffers from isoform A (SEQ ID NO:49) in that it is missing amino acids190-214 (i.e. deletion of amino acids 190-214 of SEQ ID NO:49). IsoformC is shown in SEQ ID NO:51 and differs from isoform A (SEQ ID NO:49) inthat it is missing amino acids 146-189 (i.e. deletion of amino acids146-189 of (SEQ ID NO:49). Isoform D is shown in SEQ ID NO:52 anddiffers from isoform A (SEQ ID NO:49) in that it is missing amino acids146-189 and 190-214 (i.e. deletion of amino acids 146-189 and 190-214 ofSEQ ID NO:49).

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The terms “neurological disorder” or “neurological disease” refer to ordescribe a disease or disorder of the central and/or peripheral nervoussystem in mammals. Examples of neurological disorders include, but arenot limited to the following list of disease and disorders. Neuropathydisorders are diseases or abnormalities of the nervous systemcharacterized by inappropriate or uncontrolled nerve signaling or lackthereof, and include, but are not limited to, chronic pain (includingnociceptive pain (pain caused by an injury to body tissues, includingcancer-related pain), neuropathic pain (pain caused by abnormalities inthe nerves, spinal cord, or brain), and psychogenic pain (entirely ormostly related to a psychological disorder), headache, migraine,neuropathy, and symptoms and syndromes often accompanying suchneuropathy disorders such as vertigo or nausea. Amyloidoses are a groupof diseases and disorders associated with extracellular proteinaceousdeposits in the CNS, including, but not limited to, secondaryamyloidosis, age-related amyloidosis, Alzheimer's Disease (AD), mildcognitive impairment (MCI), Lewy body dementia, Down's syndrome,hereditary cerebral hemorrhage with amyloidosis (Dutch type); the GuamParkinson-Dementia complex, cerebral amyloid angiopathy, Huntington'sdisease, progressive supranuclear palsy, multiple sclerosis; CreutzfeldJacob disease, Parkinson's disease, transmissible spongiformencephalopathy, HIV-related dementia, amyotropic lateral sclerosis(ALS), inclusion-body myositis (IBM), and ocular diseases relating tobeta-amyloid deposition (i.e., macular degeneration, drusen-relatedoptic neuropathy, and cataract). Cancers of the CNS are characterized byaberrant proliferation of one or more CNS cell (i.e., a neural cell) andinclude, but are not limited to, glioma, glioblastoma multiforme,meningioma, astrocytoma, acoustic neuroma, chondroma, oligodendroglioma,medulloblastomas, ganglioglioma, Schwannoma, neurofibroma,neuroblastoma, and extradural, intramedullary or intradural tumors.Ocular diseases or disorders are diseases or disorders of the eye, whichfor the purposes herein is considered a CNS organ subject to the BBB.Ocular diseases or disorders include, but are not limited to, disordersof sclera, cornea, iris and ciliary body (i.e., scleritis, keratitis,corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson'ssuperficial punctate keratopathy, corneal neovascularisation, Fuchs'dystrophy, keratoconus, keratoconjunctivitis sicca, iritis and uveitis),disorders of the lens (i.e., cataract), disorders of choroid and retina(i.e., retinal detachment, retinoschisis, hypertensive retinopathy,diabetic retinopathy, retinopathy, retinopathy of prematurity,age-related macular degeneration, macular degeneration (wet or dry),epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma,floaters, disorders of optic nerve and visual pathways (i.e., Leber'shereditary optic neuropathy and optic disc drusen), disorders of ocularmuscles/binocular movement accommodation/refraction (i.e., strabismus,ophthalmoparesis, progressive external opthalmoplegia, esotropia,exotropia, hypermetropia, myopia, astigmatism, anisometropia, presbyopiaand ophthalmoplegia), visual disturbances and blindness (i.e.,amblyopia, Lever's congenital amaurosis, scotoma, color blindness,achromatopsia, nyctalopia, blindness, river blindness andmicro-opthalmia/coloboma), red eye, Argyll Robertson pupil,keratomycosis, xerophthalmia and andaniridia. Viral or microbialinfections of the CNS include, but are not limited to, infections byviruses (i.e., influenza, HIV, poliovirus, rubella,), bacteria (i.e.,Neisseria sp., Streptococcus sp., Pseudomonas sp., Proteus sp., E. coli,S. aureus, Pneumococcus sp., Meningococcus sp., Haemophilus sp., andMycobacterium tuberculosis) and other microorganisms such as fungi(i.e., yeast, Cryptococcus neoformans), parasites (i.e., toxoplasmagondii) or amoebas resulting in CNS pathophysiologies including, but notlimited to, meningitis, encephalitis, myelitis, vasculitis and abscess,which can be acute or chronic. Inflammation of the CNS is inflammationthat is caused by an injury to the CNS, which can be a physical injury(i.e., due to accident, surgery, brain trauma, spinal cord injury,concussion) or an injury due to or related to one or more other diseasesor disorders of the CNS (i.e., abscess, cancer, viral or microbialinfection). Ischemia of the CNS, as used herein, refers to a group ofdisorders relating to aberrant blood flow or vascular behavior in thebrain or the causes therefor, and includes, but is not limited to, focalbrain ischemia, global brain ischemia, stroke (i.e., subarachnoidhemorrhage and intracerebral hemorrhage), and aneurysm.Neurodegenerative diseases are a group of diseases and disordersassociated with neural cell loss of function or death in the CNS, andinclude, but are not limited to, adrenoleukodystrophy, Alexander'sdisease, Alper's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, cockayne syndrome, corticobasaldegeneration, degeneration caused by or associated with an amyloidosis,Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy'sdisease, multiple system atrophy, multiple sclerosis, primary lateralsclerosis, progressive supranuclear palsy, spinal muscular atrophy,transverse myelitis, Refsum's disease, and spinocerebellar ataxia.Seizure diseases and disorders of the CNS involve inappropriate and/orabnormal electrical conduction in the CNS, and include, but are notlimited to, epilepsy (i.e., absence seizures, atonic seizures, benignRolandic epilepsy, childhood absence, clonic seizures, complex partialseizures, frontal lobe epilepsy, febrile seizures, infantile spasms,juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastautsyndrome, Landau-Kleffner Syndrome, Dravet's syndrome, Otahara syndrome,West syndrome, myoclonic seizures, mitochondrial disorders, progressivemyoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen'sSyndrome, simple partial seizures, secondarily generalized seizures,temporal lobe epilepsy, toniclonic seizures, tonic seizures, psychomotorseizures, limbic epilepsy, partial-onset seizures, generalized-onsetseizures, status epilepticus, abdominal epilepsy, akinetic seizures,autonomic seizures, massive bilateral myoclonus, catamenial epilepsy,drop seizures, emotional seizures, focal seizures, gelastic seizures,Jacksonian March, Lafora Disease, motor seizures, multifocal seizures,nocturnal seizures, photosensitive seizure, pseudo seizures, sensoryseizures, subtle seizures, sylvan seizures, withdrawal seizures, andvisual reflex seizures) Behavioral disorders are disorders of the CNScharacterized by aberrant behavior on the part of the afflicted subjectand include, but are not limited to, sleep disorders (i.e., insomnia,parasomnias, night terrors, circadian rhythm sleep disorders, andnarcolepsy), mood disorders (i.e., depression, suicidal depression,anxiety, chronic affective disorders, phobias, panic attacks,obsessive-compulsive disorder, attention deficit hyperactivity disorder(ADHD), attention deficit disorder (ADD), chronic fatigue syndrome,agoraphobia, post-traumatic stress disorder, bipolar disorder), eatingdisorders (i.e., anorexia or bulimia), psychoses, developmentalbehavioral disorders (i.e., autism, Rett's syndrome, Aspberger'ssyndrome), personality disorders and psychotic disorders (i.e.,schizophrenia, delusional disorder, and the like). Lysosomal storagedisorders are metabolic disorders which are in some cases associatedwith the CNS or have CNS-specific symptoms; such disorders include, butare not limited to Tay-Sachs disease, Gaucher's disease, Fabry disease,mucopolysaccharidosis (types I, II, III, IV, V, VI and VII), glycogenstorage disease, GM1-gangliosidosis, metachromatic leukodystrophy,Farber's disease, Canavan's leukodystrophy, and neuronal ceroidlipofuscinoses types 1 and 2, Niemann-Pick disease, Pompe disease, andKrabbe's disease.

II. Compositions and Methods

In one aspect, the invention is based, in part, on antibodies which bindBACE1 and reduce and/or inhibit BACE1 activity. In certain embodiments,antibodies that bind to the active site or an exosite of BACE1 areprovided.

A. Exemplary Anti-BACE1 Antibodies

In one aspect, the invention provides an anti-BACE1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, 23, 26, 28,45, 68, 71, 72, 73 or 120; (b) HVR-H2 comprising the amino acid sequenceof SEQ ID NO:24, 29, 46, 69, 74, 75, 76, 77, 78 or 121; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25, 30, 47, 70, 79 or122; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:7, 8,17, 35 or 42; (e) HVR-L2 comprising the amino acid sequence of SEQ IDNO:9, 10, 18, 36-39, 41, 43, 56, 58, 59, 60, 61, 62, 63, 64 or 118; and(f) HVR-L3 comprising the amino acid sequence of SEQ ID NO:11-16, 19,40, 44, 57, 65, 66, 67 or 119.

In one aspect, the invention provides an antibody comprising at leastone, at least two, or all three VH HVR sequences selected from (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO: 22, 23, 26, 28,45, 68, 71-73 or 120; (b) HVR-H2 comprising the amino acid sequence ofSEQ ID NO: 24, 29, 46, 69, 74-78 or 121; and (c) HVR-H3 comprising theamino acid sequence of SEQ ID NO: 25, 30, 47, 70, 79 or 122.

In one embodiment, the antibody comprises HVR-H1 comprising the aminoacid sequence SEQ ID NO:22 or SEQ ID NO:23 or SEQ ID NO:28 or SEQ IDNO:71 or SEQ ID NO:72 or SEQ ID NO:73. In another embodiment, theantibody comprises HVR-H2 comprising the amino acid sequence SEQ IDNO:24 or SEQ ID NO:29 or SEQ ID NO:74 or SEQ ID NO:75 or SEQ ID NO:76 orSEQ ID NO:77 or SEQ ID NO:78. In an additional embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence SEQ ID NO:25 or SEQID NO:30 or SEQ ID NO:79. In one embodiment, the antibody comprisesHVR-H1 comprising the amino acid sequence SEQ ID NO:28. In anotherembodiment, the antibody comprises HVR-H2 comprising the amino acidsequence SEQ ID NO:29. In an additional embodiment, the antibodycomprises HVR-H3 comprising the amino acid sequence SEQ ID NO:30.

In another embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:22; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:24; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:23; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:24; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25. In an additional embodiment, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:28;(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:29; and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:30. In anadditional embodiment, the antibody comprises (a) HVR-H1 comprising theamino acid sequence of SEQ ID NO:23; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:74; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:23; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:75; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:71; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:24; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:72; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:24; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:23; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:76; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:23; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:77; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:79 or the antibody comprises (a) HVR-H1 comprisingthe amino acid sequence of SEQ ID NO:73; (b) HVR-H2 comprising the aminoacid sequence of SEQ ID NO:78; and (c) HVR-H3 comprising the amino acidsequence of SEQ ID NO:25.

In another aspect, the invention provides an antibody comprising atleast one, at least two, or all three VL HVR sequences selected from (a)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 7, 8, 17, 35 and42; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 9, 10,18, 36-39, 41, 43, 56, 58-64 or 118; and (c) HVR-L3 comprising the aminoacid sequence of SEQ ID NO: 11-16, 19, 40, 44, 57, 65-67 or 119.

In one embodiment, the antibody comprises HVR-L1 comprising the aminoacid sequence SEQ ID NO:7 or SEQ ID NO:8. In another embodiment, theantibody comprises HVR-L2 comprising the amino acid sequence SEQ ID NO:9or SEQ ID NO:10 or SEQ ID NO:58 or SEQ ID NO:59 or SEQ ID NO:60 or SEQID NO:61 or SEQ ID NO:62 or SEQ ID NO:63 or SEQ ID NO:64. In anadditional embodiment, the antibody comprises HVR-L3 comprising theamino acid sequence of SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID NO:13 orSEQ ID NO:14 or SEQ ID NO:15 or SEQ ID NO:16 or SEQ ID NO:65 or SEQ IDNO:66 or SEQ ID NO:67. In another embodiment, the antibody comprisesHVR-L1 comprising the amino acid sequence SEQ ID NO:35. In anotherembodiment, the antibody comprises HVR-L2 comprising the amino acidsequence selected from the group consisting of SEQ ID NO:36-39. In anadditional embodiment, the antibody comprises HVR-L3 comprising theamino acid sequence SEQ ID NO:40.

In another embodiment, the antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:11 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:13 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:14 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:16 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:8; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:10; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:15. In an additional embodiment, the antibodycomprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:35;(b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:36; and (c)HVR-L3 comprising the amino acid sequence of SEQ ID NO:40 or theantibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQID NO:35; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:37;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:40 or theantibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQID NO:35; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:38;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:40 or theantibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQID NO:35; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39;and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:40.

In another embodiment, the antibody comprises (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO:58; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:65 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:59; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:66 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:9; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:67 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:60; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:67 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:61; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:65 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:59; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:66 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:62; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:67 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:63; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12 or the antibody comprises (a) HVR-L1 comprisingthe amino acid sequence of SEQ ID NO:7; (b) HVR-L2 comprising the aminoacid sequence of SEQ ID NO:64; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO:12.

In another aspect, an antibody of the invention comprises (a) a VHdomain comprising at least one, at least two, or all three VH HVRsequences selected from (i) HVR-H1 comprising the amino acid sequenceselected from SEQ ID NO:22, 23, 26, 28, 45, 68, 71-73 or 120 (ii) HVR-H2comprising the amino acid sequence selected from SEQ ID NO: 24, 29, 46,69, 74-78 or 121 and (iii) HVR-H3 comprising an amino acid sequenceselected from SEQ ID NO: 25, 30, 47, 70, 79 or 122; and (b) a VL domaincomprising at least one, at least two, or all three VL HVR sequencesselected from (i) HVR-L1 comprising the amino acid sequence selectedfrom SEQ ID NO: 7, 8, 17, 35 or 42, (ii) HVR-L2 comprising the aminoacid sequence selected from SEQ ID NO: 9, 10, 18, 36-39, 41, 43, 56,58-64 or 118, and (c) HVR-L3 comprising the amino acid sequence selectedfrom SEQ ID NO: 11-16, 19, 40, 44, 57, 65-67 or 119.

In another aspect, the invention provides an antibody comprising (a)HVR-H1 comprising the amino acid sequence of SEQ ID NO:23; (b) HVR-H2comprising the amino acid sequence of SEQ ID NO:24; (c) HVR-H3comprising the amino acid sequence of SEQ ID NO:25; (d) HVR-L1comprising the amino acid sequence of SEQ ID NO:7; (e) HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:9; and (f) HVR-L3 comprising anamino acid sequence selected from SEQ ID NO:12.

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the aminoacid sequence GFX₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO:45), wherein X₃₀=N or T;X₃₁=S, L or Y; X₃₂=G or Y; X₃₃=Y or S; and X₃₄=A, G or S; wherein HVR-H2comprises the amino acid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG (SEQ IDNO:46), wherein X₃₅=A or G; X₃₆=W or S; X₃₇=A or Y; X₃₈=G or S; X₃₉=S orY; and X₄₀=D or S; and wherein HVR-H3 comprises the sequenceX₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO:47), wherein X₄₁=Q or G; X₄₂=T orF; X₄₃=H or S; X₄₄=Y or P; X₄₅=Y or W; X₄₆=Y or V and wherein X₄₇optionally includes the sequence YAKGYKA (SEQ ID NO:48).

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises theamino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO:120), whereinX₇₁=F or Y; X₇₂=F, N or T; X₇₃=F or Y; X₇₄=L, Q, I, S or Y; X₇₅=G or Y;X₇₆=Y or S; and X₇₇=A, G or S; HVR-H2 comprises the amino acid sequenceX₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO:121), wherein X₇₈=A or G;X₇₉=W or S; X₈₀=A, S, Q or Y; X₈₁=G or S; X₈₂=S, K, L or Y; X₈₃=T or Y;and X₈₄=D or S; and HVR-H3 comprises the amino acid sequenceX₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO:122), wherein X₈₅=Q or G; X₈₆=T orF; X₈₇=H, Y or S; X₈₈=Y or P; X₈₉=Y or W; X₉₀=Y or V and wherein X₉₁optionally includes the sequence YAKGYKA (SEQ ID NO:48).

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A, (SEQ ID NO:42) wherein X₁₇=S, D orV; X₁₈=S or A; X₁₉=S, T or N; X₂₀=A or S; X₂₁=V or L, wherein HVR-L2comprises the amino acid sequence X₂₂ASX₂₃LYS (SEQ ID NO:43), whereinX₂₂=S, W, Y or L; X₂₃=F, S or W, and wherein HVR-L3 comprises the aminoacid sequence QQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T (SEQ ID NO:44), wherein X₂₄=S, F, G,D or Y; X₂₅=Y, P, S or A; X₂₆=Y, T or N; X₂₇=T, Y, D or S; X₂₈=P or L;and X₂₉=F, P or T.

In certain embodiments, the antibody comprises at least one sequenceselected from the group of HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1comprises the amino acid sequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:42),wherein X₁₇=S, D or V; X₁₈=S or A; X₁₉=S, T or N; X₂₀=A or S; X₂₁=V orL, wherein HVR-L2 comprises the amino acid sequence X₆₂ASX₆₃X₆₄YX₆₅ (SEQID NO:118), wherein X₆₂=S, W, Y, F or L; X₆₃=F, S, Y or W; X₆₄=L or R;X₆₅=S, P, R, K or W, and HVR-L3 comprises the amino acid sequenceQQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ ID NO:119), wherein X₆₆=S, F, G, D or Y;X₆₇=Y, P, S or A; X₆₈=Y, T or N; X₆₉=T, Y, D or S; X₇₀=P, Q, S, K or L;and X₇₁=F, P or T.

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V; X₂=Sor A; X₃=T or N; X₄=S or A; X₅=V or L, wherein HVR-L2 comprises theamino acid sequence X₆ASFLYS (SEQ ID NO:18), wherein X₆=S or L, andwherein the HVR-L3 comprises the amino acid sequence QQX₇X₈X₉X₁₀X₁₁X₁₂T(SEQ ID NO:19), wherein X₇=S, F, G, D or Y; X₈=Y, P, S, or A; X₉=T or N;X₁₀=T, Y, D or S; X₁₁=P or L; X₁₂=P or T.

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V; X₂=Sor A; X₃=T or N; X₄=S or A; X₅=V or L, wherein HVR-L2 comprises theamino acid sequence X₄₈ASX₄₉X₅₀YX₅₁ (SEQ ID NO:56), wherein X₄₈=S or F;X₄₉=F or Y; X₅₀=L or R; X₅₁=S, P, R, K or W, wherein HVR-L3 comprisesthe amino acid sequence QQFPTYX₅₂PT (SEQ ID NO:57), wherein X₅₂=L, Q, Sor K.

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the aminoacid sequence GFTFX₁₃GYX₁₄IH (SEQ ID NO:26), wherein X₁₃=S or L andX₁₄=A or G, wherein the HVR-H2 comprises the amino acid sequenceGWISPAGGSTDYADSVKG (SEQ ID NO:24), and wherein the HVR-H3 comprises theamino acid sequence GPFSPWVMDY (SEQ ID NO:25).

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-H1, HVR-H2, HVR-H3, wherein HVR-H1 comprises the aminoacid sequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO:68), wherein X₅₃=F or Y;X₅₄=T or F; X₅₅=F or Y; X₅₆=L, Q or I, wherein HVR-H2 comprises theamino acid sequence GWISPX₅₇X₅₈GX₅₉X₆₀DYADSVKG (SEQ ID NO:69), whereinX₅₇=A, S or Q; X₅₈=G or S; X₅₉=S, K or L; X₆₀=T or Y, and wherein theHVR-H3 sequence comprises the amino acid sequence GPFX₆₁PWVMDY (SEQ IDNO:70), wherein X₆₁=S or Y.

In certain embodiments, the antibody comprises at least one sequenceselected from HVR-L1, HVR-L2, HVR-L3, wherein HVR-L1 comprises the aminoacid sequence RASQSVSSAVA (SEQ ID NO:35), wherein HVR-L2 comprises theamino acid sequence X₁₅ASX₁₆LYS (SEQ ID NO:41), wherein X₁₅=S, W or Yand X₁₆=S or W, and wherein HVR-L3 comprises the amino acid sequenceQQYSYSPFT (SEQ ID NO:40).

In certain embodiments, any one or more amino acids of an anti-BACE1antibody, as provided, above are substituted at the following HVRpositions:

in HVR-H1 (SEQ ID NO:26): positions 5 and 8;

in HVR-L1 (SEQ ID NO:17): positions 5, 7, 8, 9 and 10;

in HVR-L2 (SEQ ID NO:18): position 1 or HVR-L2 (SEQ ID NO:41) positions1 and 4; and

in HVR-L3 (SEQ ID NO:19): positions 3, 4, 5, 6, 7 and 8.

In certain embodiments, the substitutions are conservativesubstitutions, as provided herein. In certain embodiments, any one ormore of the following substitutions may be made in any combination:

in HVR-H1 (SEQ ID NO:26): serine or leucine at position 5 and alanine orglycine at position 8;

in HVR-L1 (SEQ ID NO:17): aspartic acid or valine at position 5; serineor alanine at position 7; threonine or asparagine at position 8; serineor alanine at position 9 and valine or leucine at position 10;

in HVR-L2 (SEQ ID NO:18): serine or leucine at position 1 or HVR-L2 (SEQID NO:41) serine, tyrosine or tryptophan at position 1 or tyrosine,serine or tryptophan at position 4; and

in HVR-L3 (SEQ ID NO:19): serine, phenylalanine, glycine, aspartic acidor tyrosine at position 3; tyrosine or proline at position 4, serine,alanine, threonine or asparagine at position 5; tyrosine, threonine,aspartic acid or serine at position 6, aspartic acid, serine, proline orleucine at position 7 and proline or threonine at position 8.

In certain embodiments, the substitutions are conservativesubstitutions, as provided herein. In certain embodiments, any one ormore of the following substitutions may be made in any combination:

in HVR-H1 (SEQ ID NO:26): S5L and ABG;

in HVR-L1 (SEQ ID NO:17): DSV; S7A; T8N; S9A and VIOL;

in HVR-L2 (SEQ ID NO:18): S1L or HVR-L2 (SEQ ID NO:41) positions S1W orY and S4W; and

in HVR-L3 (SEQ ID NO:19): positions S3F, G, D or Yl; Y4P, S or A; T5N;T6Y, D or S; P7L and PBT.

In certain embodiments, any one or more amino acids of an anti-BACE1antibody, as provided, above are substituted at the following HVRpositions:

in HVR-H1 (SEQ ID NO:120): positions 2, 3, 5, 6, 7 and 8;

in HVR-H2 (SEQ ID NO:121): positions 1, 2, 6, 7, 9, 10, and 11;

in HVR-H3 (SEQ ID NO:122) positions 1, 3, 4, 5, 6, 7, and 8

in HVR-L1 (SEQ ID NO:42): positions 5, 7, 8, 9 and 10;

in HVR-L2 (SEQ ID NO:118): position 1, 4, 5 and 7; and

in HVR-L3 (SEQ ID NO:119): positions 3, 4, 5, 6, 7 and 8.

In certain embodiments, the substitutions are conservativesubstitutions, as provided herein.

Possible combinations of the above substitutions are encompassed by theconsensus sequences of SEQ ID NO:42-47 and 118-122 as described above.

In any of the above embodiments, an anti-BACE1 antibody is humanized. Inone embodiment, an anti-BACE1 antibody comprises HVRs as in any of theabove embodiments, and further comprises an acceptor human framework,e.g. a human immunoglobulin framework or a human consensus framework. Inanother embodiment, an anti-BACE1 antibody comprises HVRs as in any ofthe above embodiments, and further comprises a VH or VL comprising anFR1, FR2, FR3, or FR4 sequence of SEQ ID NO:1-6, 20, 21, 27, 31-34,80-98 and 99-117.

In another aspect, an anti-BACE1 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence selected from SEQ ID NO:20, 21, 27 and 80-98. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-BACE1 antibody comprising that sequenceretains the ability to bind to BACE1 and/or inhibit or reduce BACE1activity. In certain embodiments, a total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NO:20, 21, 27 and80-98. In certain embodiments, substitutions, insertions, or deletionsoccur in regions outside the HVRs (i.e., in the FRs). Optionally, theanti-BACE1 antibody comprises the VH sequence in SEQ ID NO:20, 21, 27 or80-98, including post-translational modifications of that sequence. In aparticular embodiment, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:22, 23,26, 28, 45, 68, 71, 72, 73 or 120, (b) HVR-H2 comprising the amino acidsequence of SEQ ID NO:24, 29, 46, 69, 74, 75, 76, 77, 78 or 121, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO:25, 30, 47, 70,79 or 122.

In one aspect, the invention provides an anti-BACE1 antibody comprisingat least one, two, three, four, five, or six HVRs selected from the (a)HVR-H1 comprising an amino acid sequence in FIGS. 1(B), 2(B) and 24(A);(b) HVR-H2 comprising an amino acid sequence in FIGS. 1(B), 2(B) and24(B); (c) HVR-H3 comprising an amino acid sequence in FIGS. 1(B), 2(B)and 24(C); (d) HVR-L1 comprising an amino acid sequence in FIGS. 1(A),2(A) and 23(A); (e) HVR-L2 comprising an amino acid sequence in FIGS.1(A), 2(A) and 23(B); and (f) HVR-L3 comprising an amino acid sequencein FIGS. 1(A) and 2(A) and 23(C).

In another aspect, an anti-BACE1 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence selected from SEQ ID NO:1-6, 31-34and 99-117. In certain embodiments, a VL sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-BACE1 antibodycomprising that sequence retains the ability to bind to BACE1 and/orinhibit or reduce BACE1 activity. In certain embodiments, a total of 1to 10 amino acids have been substituted, inserted and/or deleted in SEQID NO:1-6, 31-34 and 99-117. In certain embodiments, the substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-BACE1 antibody comprises the VL sequence inSEQ ID NO:1-6, 31-34 or 99-117, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three HVRs selected from (a) HVR-L1 comprising theamino acid sequence of SEQ ID NO:7, 8, 17, 35 or 42; (b) HVR-L2comprising the amino acid sequence of SEQ ID NO:9, 10, 18, 36-39, 41, 43and 56, 58-64 or 118; and (c) HVR-L3 comprising the amino acid sequenceof SEQ ID NO:11-16, 19, 40, 44, 57, 65, 66, 67 or 119.

In another aspect, an anti-BACE1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises the VH and VL sequences in SEQ ID NO:21 and SEQ IDNO:2, respectively, including post-translational modifications of thosesequences.

In a further aspect, the invention provides an antibody that binds tothe same epitope as an anti-BACE1 antibody provided herein. For example,in certain embodiments, an antibody is provided that binds to the sameepitope as an anti-BACE1 antibody comprising a VH sequence selected fromSEQ ID NO: 20, 21, 27 and 80-98 and a VL sequence selected from SEQ IDNO: 1-6, 31-34 and 99-117. In certain embodiments, an antibody isprovided that binds to the same epitope as an anti-BACE1 antibodycomprising the VH and VL sequences in SEQ ID NO: 21 and SEQ ID NO:2,respectively.

In certain embodiments, an antibody is provided that binds to an epitopewithin BACE1 comprising at least one, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine amino acid(s) which corresponds to the amino acids 314SER, 316 GLU, 317 LYS, 318 PHE, 319 PRO, 327 GLN, 328 LEU, 329 VAL, 330CYS, 331 TRP, 332 GLN, 333 ALA, 335 THR, 337 PRO, 340 ILE, 375 THR, 378ASP, 380 CYS, 426 PHE of SEQ ID NO:49.

In certain embodiments, an antibody is provided that binds to an epitopewithin BACE1 comprising at least one, at least two, at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine amino acid(s) which corresponds to the amino acids 314SER; 316 GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and378 ASP of SEQ ID NO:49. In other embodiments the conformational epitopecomprises amino acids which correspond to 314 SER; 316 GLU; 317 LYS; 327GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49. Itwill be appreciated that the amino acids identified in the BACE1 epitopecorrespond to the sequence of human BACE1 isoform A. However, thedescribed BACE1 conformational epitope also encompasses correspondingamino acids in other variants and isoforms of BACE1 and the epitope mayinclude amino acids other than the residues specified.

In certain embodiments, an antibody is provided that binds to an epitopewithin BACE1 comprising at least one, at least two or at least threeamino acid region(s) of BACE1 which correspond to amino acids 315-318 ofSEQ ID NO:49; amino acids 331-335 of SEQ ID NO:49; amino acids 370-381of SEQ ID NO:49; or any combination thereof. In one embodiment, theantibody binds to an epitope of BACE1 comprising amino acids 315-318,331-335 and 370-381 of SEQ ID NO:49.

In another embodiment an antibody is provided that binds to an epitopewithin BACE1 which results in a conformational change in the P6 and/orP7 sites (Turner et al., Biochemistry 44:105-112 (2005)) of BACE1 uponbinding relative to BACE1 without the antibody bound. In an additionalembodiment, an antibody is provided that binds to an epitope of BACE1which induces amino acids 218-231 of SEQ ID NO:49 of BACE1 to adopt arandom loop structure. Amino acids 218-231 of SEQ ID NO:49 of BACE1exist in an α-helical structure in the substrate bound complex.

In another embodiment, an antibody is provided that binds to a sitewithin BACE1 as indicated in FIGS. 14 and 15 and described in thecrystal structure of BACE1 and the anti-BACE1 antibody, YW412.8.31(Example 3(B)).

In other embodiments, an antibody is provided that binds to an exositewithin BACE1. In one embodiment, the exosite within BACE1 is the sameexosite as that identified by Kornacker et al., Biochem. 44:11567-11573(2005). In one embodiment an antibody is provided that competes with thepeptides identified in Kornacker et al., Biochem. 44:11567-11573 (2005),which is incorporated herein by reference in its entirety, (i.e.,Peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7,1-6, 2-12, 3-12, 4-12, 5-12,6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9, Y5A, P6A, Y7A, F8A,I9A, P10A and L11A) for binding to BACE1.

In another embodiment, an antibody is provided that competes for binding(e.g., binds to the same epitope) as any anti-BACE1 antibody describedherein.

In a further aspect of the invention, an anti-BACE1 antibody accordingto any of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-BACE1antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype as defined herein.

In a further aspect, an anti-BACE1 antibody according to any of theabove embodiments may incorporate any of the features, singly or incombination, as described in Sections 1-7 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 μM or 26 μM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mot. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with astirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for BACE1 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of BACE1. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express BACE1. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to BACE1 as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to an Fcregion. For example, the amount of fucose in such antibody may be from1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amountof fucose is determined by calculating the average amount of fucosewithin the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e.g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed in WO 2008/077546, for example. Asn297 refers to theasparagine residue located at about position 297 in the Fc region (Eunumbering of Fc region residues); however, Asn297 may also be locatedabout ±3 amino acids upstream or downstream of position 297, i.e.,between positions 294 and 300, due to minor sequence variations inantibodies. Such fucosylation variants may have improved ADCC function.See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publicationsrelated to “defucosylated” or “fucose-deficient” antibody variantsinclude: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614;US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umanaet al.). Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Natl. Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Natl. Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Natl. Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Intl. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and 5400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-BACE1 antibody described hereinis provided. Such nucleic acid may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., expression vectors)comprising such nucleic acid are provided. In a further embodiment, ahost cell comprising such nucleic acid is provided. In one suchembodiment, a host cell comprises (e.g., has been transformed with): (1)a vector comprising a nucleic acid that encodes an amino acid sequencecomprising the VL of the antibody and an amino acid sequence comprisingthe VH of the antibody, or (2) a first vector comprising a nucleic acidthat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-BACE1 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-BACE1 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

C. Assays

Anti-BACE1 antibodies provided herein may be identified, screened for,or characterized for their physical/chemical properties and/orbiological activities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with any of the antibodies or Fabs descriedherein, for example, YW412.8, YW412.8.31, YW412.8.30, YW412.8.2,YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10 for binding to BACE1. Incertain embodiments, such a competing antibody binds to the same epitope(e.g., a linear or a conformational epitope) that is bound by any of theantibodies or Fabs descried herein, for example, YW412.8, YW412.8.31,YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized BACE1 is incubated in asolution comprising a first labeled antibody that binds to BACE1 (e.g.,YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51,Fab12, LC6, LC9, LC10) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toBACE1. The second antibody may be present in a hybridoma supernatant. Asa control, immobilized BACE1 is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto BACE1, excess unbound antibody is removed, and the amount of labelassociated with immobilized BACE1 is measured. If the amount of labelassociated with immobilized BACE1 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toBACE1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-BACE1 antibodiesthereof having biological activity. Biological activity may include,e.g., inhibition or reduction of BACE1 aspartyl protease activity; orinhibition or reduction in APP cleavage by BACE1; or inhibition orreduction in Aβ production. Antibodies having such biological activityin vivo and/or in vitro are also provided.

In certain embodiments, an antibody of the invention is tested for suchbiological activity. For example, BACE1 protease activity can be testedin an homogeneous time-resolved fluorescence HTRF assay or amicrofluidic capillary electrophoretic (MCE) assay, as described indetail in Example 1 and 2(B), using synthetic substrate peptides.

Briefly, a homogeneous time-resolved fluorescence (HTRF) assay can beused to measure BACE1 aspartyl protease activity with the use of anamyloid precursor protein BACE1 cleavage site peptide. For example, theBi27 peptide (Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO:53),American Peptide Company)), is combined with BACE1 pre-incubated with ananti-BACE antibody in BACE reaction buffer (50 mM sodium acetate pH 4.4and 0.1% CHAPS) in a 384-well plate (Proxiplate™, Perkin-Elmer). Theproteolytic reaction mixture is incubated at ambient temperature for 75minutes and was quenched by the addition of 5 μL HTRF detection mixturecontaining 2 nM Streptavidin-D2 and 150 nM of an anti-amyloid betaantibody labeled with Europium cryptate in detection buffer (200 mM TrispH 8.0, 20 mM EDTA, 0.1% BSA, and 0.8M KF). The final reaction mixtureis incubated at ambient temperature for 60 minutes and the TR-FRETsignal is measured using an EnVision Multilabel Plate Reader™(Perkin-Elmer) at an excitation wavelength of 320 nm and emissionwavelengths of 615 and 665 nm.

An MCE assay reactions can be carried out in a standard enzymaticreaction, initiated by the addition of substrate to enzyme and 4×compound, containing human BACE1 (extracellular domain), amyloidprecursor protein beta secretase active site peptide(FAM-KTEEISEVNLDAEFRWKK-CONH₂ (SEQ ID NO:55)), 50 mM NaOAc pH 4.4 and0.1% CHAPS. After incubation for 60 minutes at ambient temperature, theproduct and substrate in each reaction is separated using a 12-sippermicrofluidic chip analyzed on an LC3000® (both, Caliper Life Sciences).The separation of product and substrate is optimized by choosingvoltages and pressure using the manufacturer's optimization software.Substrate conversion is calculated from the electrophoregram using HTSWell Analyzer software (Caliper Life Sciences).

In addition, BACE1 protease activity can be tested in vivo in cell lineswhich express BACE1 substrates such as APP, or in transgenic mice whichexpress BACE1 substrates, such as human APP, as described in Examples2(C) and 4.

Additionally, BACE1 protease activity can be tested with anti-BACE1antibodies in animal models. For example, animal models of variousneurological diseases and disorders, and associated techniques forexamining the pathological processes associated with these models, arereadily available in the art. Animal models of various neurologicaldisorders include both non-recombinant and recombinant (transgenic)animals. Non-recombinant animal models include, for example, rodent,e.g., murine models. Such models can be generated by introducing cellsinto syngeneic mice using standard techniques, e.g. subcutaneousinjection, tail vein injection, spleen implantation, intraperitonealimplantation, and implantation under the renal capsule. In vivo modelsinclude models of stroke/cerebral ischemia, in vivo models ofneurodegenerative diseases, such as mouse models of Parkinson's disease;mouse models of Alzheimer's disease; mouse models of amyotrophic lateralsclerosis; mouse models of spinal muscular atrophy; mouse/rat models offocal and global cerebral ischemia, for instance, common carotid arteryocclusion or middle cerebral artery occlusion models; or in ex vivowhole embryo cultures. As one nonlimiting example, there are a number ofart-known mouse models for Alzheimer's disease ((see, e.g. Rakover etal., Neurodegener. Dis. (2007); 4(5): 392-402; Mouri et al., FASEB J.(2007) July; 21 (9): 2135-48; Minkeviciene et al., J. Pharmacol. Exp.Ther. (2004) November; 311 (2):677-82 and Yuede et al., Behav Pharmacol.(2007) September; 18 (5-6): 347-63). The various assays may be conductedin known in vitro or in vivo assay formats, as known in the art anddescribed in the literature. Various such animal models are alsoavailable from commercial vendors such as the Jackson Laboratory.Additional animal model assays are described in Examples 4 and 5.

D. Immunoconjugates

The invention also provides immunoconjugates comprising an anti-BACE1antibody herein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med. Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med.Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-BACE1 antibodies provided hereinis useful for detecting the presence of BACE1 in a biological sample.The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as serum, plasma, saliva, gastricsecretions, mucus, cerebrospinal fluid, lymphatic fluid, neuronaltissue, brain tissue, cardiac tissue or vascular tissue.

In one embodiment, an anti-BACE1 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of BACE1 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-BACE1 antibody as described herein under conditionspermissive for binding of the anti-BACE1 antibody to BACE1, anddetecting whether a complex is formed between the anti-BACE1 antibodyand BACE1. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-BACE1 antibody is used to select subjects eligiblefor therapy with an anti-BACE1 antibody, e.g. where BACE1 is a biomarkerfor selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include neurodegenerative diseases (including, but not limitedto, Lewy body disease, postpoliomyelitis syndrome, Shy-Draeger syndrome,olivopontocerebellar atrophy, Parkinson's disease, multiple systematrophy, striatonigral degeneration, tauopathies (including, but notlimited to, Alzheimer disease and supranuclear palsy), prion diseases(including, but not limited to, bovine spongiform encephalopathy,scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), stroke, muscular dystrophy, multiplesclerosis, Amyotrophic lateral sclerosis (ALS), Angelman's syndrome,Liddle syndrome, Paget's syndrome, traumatic brain injury, bulbar palsy,motor neuron disease, and nervous system heterodegenerative disorders(including, but not limited to, Canavan disease, Huntington's disease,neuronal ceroid-lipofuscinosis, Alexander's disease, Tourette'ssyndrome, Menkes kinky hair syndrome, Cockayne syndrome,Halervorden-Spatz syndrome, lafora disease, Rett syndrome,hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome), dementia (including, but not limited to,Pick's disease, and spinocerebellar ataxia).

In certain embodiments, labeled anti-BACE1 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-BACE1 antibody as describedherein are prepared by mixing such antibody having the desired degree ofpurity with one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Such active ingredients are suitably present in combination inamounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

G. Therapeutic Methods and Compositions

Any of the anti-BACE1 antibodies provided herein may be used intherapeutic methods.

In one aspect, an anti-BACE1 antibody for use as a medicament isprovided. In further aspects, an anti-BACE1 antibody for use in treatinga neurological disease or disorder is provided (e.g., AD). In certainembodiments, an anti-BACE1 antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides an anti-BACE1antibody for use in a method of treating an individual having aneurological disease or disorder comprising administering to theindividual an effective amount of the anti-BACE1 antibody. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. Infurther embodiments, the invention provides an anti-BACE1 antibody foruse in reducing or inhibiting amyloid plaque formation in a patient atrisk or suffering from a neurological disease or disorder (e.g., AD). Incertain embodiments, the invention provides an anti-BACE1 antibody foruse in a method of reducing or inhibiting Aβ production in an individualcomprising administering to the individual an effective of theanti-BACE1 antibody. An “individual” according to any of the aboveembodiments is preferably a human. In certain aspect, the anti-BACEantibody for use in the methods of the invention reduces or inhibitsBACE1 activity. For example, the anti-BACE1 antibody reduces or inhibitsthe ability of BACE1 to cleave APP.

In a further aspect, the invention provides for the use of an anti-BACE1antibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of neurological disease ordisorder. In a further embodiment, the medicament is for use in a methodof treating neurological disease or disorder comprising administering toan individual having neurological disease or disorder an effectiveamount of the medicament. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent, e.g., as described below. In afurther embodiment, the medicament is for inhibiting BACE1 activity. Ina further embodiment, the medicament is for use in a method ofinhibiting Aβ production or plaque formation in an individual comprisingadministering to the individual an amount effective of the medicament toinhibit Aβ production or plaque formation. An “individual” according toany of the above embodiments may be a human.

In a further aspect, the invention provides a method for treatingAlzheimer's disease. In one embodiment, the method comprisesadministering to an individual having AD an effective amount of ananti-BACE1 antibody. In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent. An “individual” according to anyof the above embodiments may be a human.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-BACE1 antibodies provided herein, e.g., foruse in any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-BACE1 antibodiesprovided herein and a pharmaceutically acceptable carrier. In anotherembodiment, a pharmaceutical formulation comprises any of the anti-BACE1antibodies provided herein and at least one additional therapeuticagent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy.

An antibody of the invention (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Certain embodiments of the invention provide for the antibody orfragment thereof to traverse the blood-brain barrier. Certainneurodegenerative diseases are associated with an increase inpermeability of the blood-brain barrier, such that the antibody oractive fragment thereof can be readily introduced to the brain. When theblood-brain barrier remains intact, several art-known approaches existfor transporting molecules across it, including, but not limited to,physical methods, lipid-based methods, and receptor and channel-basedmethods.

Physical methods of transporting the antibody or fragment thereof acrossthe blood-brain barrier include, but are not limited to, circumventingthe blood-brain barrier entirely, or by creating openings in theblood-brain barrier. Circumvention methods include, but are not limitedto, direct injection into the brain (see e.g., Papanastassiou et al.,Gene Therapy 9: 398-406 (2002)) and implanting a delivery device in thebrain (see e.g., Gill et al., Nature Med. 9: 589-595 (2003); and GliadelWafers™, Guildford Pharmaceutical). Methods of creating openings in thebarrier include, but are not limited to, ultrasound (see e.g., U.S.Patent Publication No. 2002/0038086), osmotic pressure (e.g., byadministration of hypertonic mannitol (Neuwelt, E. A., Implication ofthe Blood-Brain Barrier and its Manipulation, Vols 1 & 2, Plenum Press,N.Y. (1989))), permeabilization by, e.g., bradykinin or permeabilizerA-7 (see e.g., U.S. Pat. Nos. 5,112,596, 5,268,164, 5,506,206, and5,686,416), and transfection of neurons that straddle the blood-brainbarrier with vectors containing genes encoding the antibody or fragmentthereof (see e.g., U.S. Patent Publication No. 2003/0083299).

Lipid-based methods of transporting the antibody or fragment thereofacross the blood-brain barrier include, but are not limited to,encapsulating the antibody or fragment thereof in liposomes that arecoupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody oractive fragment thereof in low-density lipoprotein particles (see e.g.,U.S. Patent Application Publication No. 20040204354) or apolipoprotein E(see e.g., U.S. Patent Application Publication No. 20040131692).

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-BACE1 antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-BACE1 antibody.

III. Examples

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1 Generation and Characterization of Anti-BACE1 Antibodies

Antibodies specifically binding to BACE1 were generated by panning twodifferent types of phage display antibody libraries (one with naturaldiversity (VH and VH/VL), the other with diversity in certain CDRregions artificially restricted to particular amino acid sets (YSGX))against the human BACE1 extracellular domain, amino acids 1-457 of SEQID NO:49.

A. Natural Diversity Library Sorting and Screening to IdentifyAnti-BACE-1 Antibodies

Selection of Phage Displayed Anti-BACE1 Clones

Biotinylated human BACE-1 (1-457 of SEQ ID NO:49) was used as an antigenfor library sorting. The natural diversity phage libraries were sortedfive rounds against biotinylated BACE-1 pre-captured on alternatingneutravidin/streptavidin plates. For the first round of sorting, NUNC 96well Maxisorp immunoplates were first coated with 10 μg/mL neutravidin(Fisher Scientific, #21125) and blocked with phage blocking buffer PBST(phosphate-buffered saline (PBS) and 1% (w/v) bovine serum albumin (BSA)and 0.05% (v/v) Tween 20) overnight. First, 10 μg/mL biotinylated BACE-1was captured on the immunoplates for 30 minutes. Antibody phagelibraries VH (see e.g., Lee et al., J. Immunol. Meth. 284:119-132(2004)) and VH/VL (see Liang et al., J. Mol. Biol. 366: 815-829 (2007)),pre-blocked with phage blocking buffer PBST, were subsequently added tothe plates and incubated overnight at room temperature. The plates werewashed 10× the following day with PBT (PBS with 0.05% Tween 20), andbound phage were eluted with 1 mL 50 mM HCl and 500 mM NaCl for 30 minand neutralized with 600 μL of 1 M Tris base (pH 8.0). Recovered phagewere amplified in E. coli XL-1 Blue cells. During the subsequentselection rounds, propagated phage libraries were first pre-absorbedwith 50 μl of Dynabeads® MyOne™ Streptavidin T1 (Invitrogen, #65601) inPBST/BSA and incubated for 30 minutes at room temperature. Phageparticles that bound to neutravidin were subtracted from the phage stockwith the removal of the Dynabeads®. The unbound phage were then added toBACE-1 antigen displayed on streptavidin plates and the incubation timewas reduced to 2-3 hours. The stringency of plate washing was graduallyincreased.

After 5 rounds of panning, significant enrichment was observed. 96clones were picked from the VH and VH/VL library sorting to determinewhether they specifically bound to human BACE-1. The variable regions ofthese clones were PCR sequenced to identify unique sequence clones. 42unique phage antibodies that bound human BACE-1 with at least 5× abovebackground were chosen and reformatted to full length IgGs forevaluation in in vitro cell assays.

Clones of interest were reformatted into IgGs by cloning V_(L) and V_(H)regions of individual clones into the LPG3 and LPG4 vectors,respectively, and transiently expressed in mammalian CHO cells, andpurified using protein A column chromatography.

Selection of Anti-BACE1 Inhibitory Clones

BACE1 is an aspartyl protease that normally cleaves amyloid precursorprotein at a point near its transmembrane domain, close to the surfaceof the cell. Accordingly, the ability of antibodies identified above tomodulate BACE1 proteolytic activity on certain BACE1 substrates, wasassessed in vitro using a homogeneous time-resolved fluorescence (HTRF)assay.

The HTRF assay was performed as follows. Two microliters of 375 nM Bi27(Biotin-KTEEISEVNLDAEFRHDSGYEVHHQKL (SEQ ID NO:53), American PeptideCompany)), an amyloid precursor protein BACE1 cleavage site peptidebearing a substitution to increase sensitivity to BACE1 cleavage, wascombined with 3 μL of 125 nM BACE1 pre-incubated with an anti-BACEantibody in BACE reaction buffer (50 mM sodium acetate pH 4.4 and 0.1%CHAPS) in a 384-well plate (Proxiplate™, Perkin-Elmer). The proteolyticreaction mixture was incubated at ambient temperature for 75 minutes andwas quenched by the addition of 5 μL HTRF detection mixture containing 2nM Streptavidin-D2 and 150 nM of 6E10 anti-amyloid beta antibody(Covance, Emoryville, Calif.) labeled with Europium cryptate indetection buffer (200 mM Tris pH 8.0, 20 mM EDTA, 0.1% BSA, and 0.8MKF). The final reaction mixture was incubated at ambient temperature for60 minutes and the TR-FRET signal was measured using an EnVisionMultilabel Plate Reader™ (Perkin-Elmer) at an excitation wavelength of320 nm and emission wavelengths of 615 and 665 nm. Reactions lackingBACE1 enzyme (0 BACE) and reactions containing lacking anti-BACE1antibodies (PBS (100% BACE1 Activity) were used as controls.

Of the 42 antibodies tested which were identified from the naturaldiversity library, the best inhibitor of BACE1, YW412.8, was chosen foraffinity maturation. See FIG. 4.

Affinity Maturation of Anti-BACE1 Inhibitory Clones

Libraries were constructed to affinity mature the YW412.8 antibody asfollows. Phagemid pW0703 (derived from phagemid pV0350-2b (Lee et al.,J. Mol. Biol. 340, 1073-1093 (2004)), containing a stop codon (TAA) inall CDR-L3 positions and displaying monovalent Fab on the surface of M13bacteriophage) served as the library template for grafting heavy chainvariable domains (VH) of clones of interest from the natural diversitylibrary for affinity maturation. Both hard and soft randomizationstrategies were used for affinity maturation. For hard randomization,one light chain library with selected positions of the three light chainCDRs was randomized using amino acids designed to mimic natural humanantibodies and the designed DNA degeneracy was as described in Lee etal. (J. Mol. Biol. 340, 1073-1093 (2004)). For soft randomization,residues at positions 91-94, and 96 of CDR-L3, 28-31 and 34-35 ofCDR-H1, 50, 52, and 53-58 of CDR-H2, 95-99 and 100A of CDR-H3, weretargeted; and two different combinations of CDR loops, L3/H1/H2 andL3/H3, were selected for randomization. To achieve the softrandomization conditions, which introduced the mutation rate ofapproximately 50% at the selected positions, the mutagenic DNA wassynthesized with 70-10-10-10 mixtures of bases favoring the wild typenucleotides (Gallop et al., J. Med. Chem. 37:1233-1251 (1994)).

Selection of affinity improved Fabs was performed as follows. Affinityimprovement phage libraries were subjected to plate sorting for thefirst round, followed by four or five rounds of solution sorting. Forthe first round of plate sorting, the libraries were sorted against 10μg/ml biotinylated target (BACE1) captured by neutravidin coated plate(NUNC Maxisorp plate) with phage input about 20D/ml in 1% BSA and 0.05%Tween 20 for 2 hours at room temperature. After the first round of platesorting, solution sorting was performed to increase the stringency ofselection. For solution sorting, 10D/ml phage propagated from the firstround of plate sorting were incubated with 100 nM biotinylated targetprotein (the concentration was based on parental clone phage IC₅₀values) in 100 μl buffer containing 1% Superblock (Pierce Biotechnology)and 0.05% Tween 20 for 30 minutes at room temperature. The mixture wasfurther diluted 10× with 1% Superblock, and 100 μl/well was applied toneutravidin-coated wells (5 μg/ml) for 15 minutes at room temperaturewith gentle shaking such that biotinylated target bound phage. The wellswere washed with PBS and 0.05% Tween 20 ten times. To determinebackground binding, control wells containing phage with targets thatwere not biotinylated were captured on neutravidin-coated plates. Boundphage were eluted with 0.1 N HCl for 20 minutes, neutralized by 1/10volume of 1 M Tris pH 11, titered, and propagated for the next round.Next, two more rounds of solution sorting were carried out together withincreasing selection stringency. The first round was for on-rateselection by decreasing biotinylated target protein concentration from100 nM to 5 nM. The second round was for off-rate selection by addingexcess amounts of non-biotinylated target protein (100-fold more) tocompete off weaker binders at room temperature. Also, the phage inputwas decreased (0.1˜0.5 OD/ml) to lower background phage binding.

Colonies were picked from the fourth round screens and were grownovernight at 37° C. in 150 μl/well of 2YT media with 50 μg/mlcarbenicillin and 1E10/ml KO7 phage in 96-well plates (Falcon). From thesame plate, a colony of XL-1 infected parental phage was picked as acontrol. 96-well Nunc Maxisorp plates were coated with 100 μl/wellneutravidin (2 μg/ml) in PBS at 4° C. overnight or room temperature for2 hours. The plates were blocked with 65 μl of 1% BSA for 30 min and 40μl of 1% Tween 20 for another 30 minutes before biotinylated targetprotein (2 μg/ml) was added and incubated for 15 min at roomtemperature.

The phage supernatant was diluted 1:10 in ELISA (enzyme linkedimmunosorbent assay) buffer (PBS with 0.5% BSA, 0.05% Tween20) with orwithout 10 nM target protein in 100 μl total volume and incubated atleast 1 hour at room temperature in an F plate (NUNC) for use in asingle spot competition assay. 75 μl of mixture with or without targetprotein was transferred side by side to the target protein captured byneutravidin-coated plates. The plate was gently shaken for 15 min toallow the capture of unbound phage to the neutravidin-captured targetprotein. The plate was washed at least five times with PBS-0.05% Tween20. The binding was quantified by adding horseradish peroxidase(HRP)-conjugated anti-M13 antibody in ELISA buffer (1:5000) andincubated for 30 minutes at room temperature. The plates were washedwith PBS-0.05% Tween 20 at least five times. Next, 100 μl/well of a 1:1ratio of 3,3′,5,5′-tetramethylbenzidine (TMB) Peroxidase substrate andPeroxidase Solution B (H₂O₂) (Kirkegaard-Perry Laboratories(Gaithersburg, Md.)) was added to the well and incubated for 5 minutesat room temperature. The reaction was stopped by adding 100 μl 1 Mphosphoric acid (H₃PO₄) to each well and allowed to incubate for 5minutes at room temperature. The OD (optical density) of the yellowcolor in each well was determined using a standard ELISA plate reader at450 nm. The OD reduction (%) was calculated by the following equation:

OD_(450 nm) reduction (%)=[(OD_(450 nm) of wells withcompetitor)/(OD_(450 nm) of well with no competitor)]*100.

In comparison to the OD_(450 nm) reduction (%) of the well of parentalphage (100%), clones that had the OD_(450 nm) reduction (%) lower than50% for both the human and murine target were picked for sequenceanalysis. Unique clones were selected for phage preparation to determinebinding affinity (phage IC₅₀) against target by comparison with parentalclones. The most affinity-improved clones were reformatted into humanIgG1 for antibody production and further binding kinetic analysis bysurface plasmon resonance using BIAcore and other in vitro or in vivoassays.

The sequence of the light chain and heavy chain HVR region of YW412.8,chosen from the natural diversity phage library, is shown in FIGS. 1(A)and 1(B). Additionally, five antibodies obtained from affinitymaturation of the YW412.8 antibody were also sequenced and the lightchain and heavy chain HVR sequences are also shown in FIGS. 1(A) and1(B). The consensus amino acid sequences of the light chain HVR regionsthat displayed variability in these antibodies were: HVR-L1: Arg Ala SerGln X₁ Val X₂ X₃ X₄ X₅ Ala (SEQ ID NO: 17), wherein X₁ is selected fromaspartic acid and valine, X₂ is selected from serine and alanine, X₃ isselected from threonine and asparagine, X₄ is selected from alanine andserine, and X₅ is selected from valine and leucine; HVR-L2: X₆ Ala SerPhe Leu Tyr Ser (SEQ ID NO: 18), wherein X₆ is selected from serine andleucine; and HVR-L3: Gln Gln X₇ X₈ X₉ X₁₀ X₁₁ X₁₂ Thr (SEQ ID NO: 19),wherein X₇ is selected from serine, phenylalanine, glycine, asparticacid and tyrosine, X₈ is selected from tyrosine, proline, serine andalanine, X₉ is selected from threonine and asparagine, X₁₀ is selectedfrom threonine, tyrosine, aspartic acid and serine, X₁₁ is selected fromproline and leucine and X₁₂ is selected from proline and threonine. Onlythe heavy chain hypervariable region H1 displayed variability amongstthese antibodies, and the consensus sequence for that region was:HVR-H1: Gly Phe Thr Phe X₁₃ Gly Tyr X₁₄ Ile His (SEQ ID NO: 26), whereinX₁₃ is selected from serine and leucine and X₁₄ is selected from alanineand glycine.

B. Synthetic Diversity Library Sorting and Screening to IdentifyAnti-BACE-1 Antibodies

Minimalist synthetic antibody libraries with restricted chemicaldiversity at the complementary determining regions (CDRs) have beenconstructed and shown to be effective in obtaining high affinityantibody binders against a variety of proteins as previously describedin Fellouse, F. A. et al. J. Mol. Biol. 373: 924-940 (2007). A syntheticdiversity library, designated as the YSGX library, was used to searchfor an inhibitory antibody against BACE1 by solution sorting. Panningfor binding was carried out for five rounds as described below.

The library for primary sorting, designated as YSGX library, wasconstructed as previously described using a phagemid for Fab-phagedisplay (pF1359) (Library D in Fellouse, F. A. et al., J. Mol. Biol.373: 924-940 (2007)). The diversity of the library was about 2×10¹⁰.

For affinity maturation, all three CDRL were randomized with fixed CDRHfor selected clones derived from the primary sorting. Three types ofoligonucleotides were used for randomization. Type I uses the degeneratecodon TMC that encodes only Tyr and Ser. Type II uses a custom trimerphosphoramidite mix containing codons for Tyr, Ser, Gly and Trp atequimolar ratios. Type III uses a trimer phosphoramidite mix encodingfor 10 amino acid residues in the following molar ratios: Tyr (30%), Ser(15%), Gly (15%), Trp (10%) and Phe, Leu, His, Asp, Pro, Ala, 5% each. Amutation was introduced into the oligonucleotides used for CDR-L3 sothat the KpnI site on the original template was silenced uponmutagenesis. The length variation was from 3 to 10 amino acids forCDR-L1, 7 amino acids for CDR-L2, and from 2-10 amino acids for CDR-L3.The oligonucleotides were pooled together properly to make the final setof oligonucleotides, i.e. mix all L1, L2 and L3 oligonucleotides withdifferent length within one type and then mix all three types togetheras the oligonucleotide set for CDR-L1, CDR-L2 and CDR-L3, respectively.Kunkel mutagenesis was used to replace all CDR-LC positions. AfterKunkel mutagenesis (Kunkel, T. A. et al., Methods Enzymol. 154: 367-382(1987)), the DNA was purified and treated with KpnI at 37° C. for 3 h todigest the template DNA. The purified DNA was then subjected toelectroporation for library construction.

Selection of Phage Displayed Anti-BACE1 Clones

Biotinylated human BACE-1 (1-457 of SEQ ID NO:49) was used as an antigenfor library sorting. For the first round of panning, 20 μg ofbiotinylated BACE1 was incubated with 1 ml of the library at aconcentration of 1×10¹³ pfu/ml at 4° C. for 1.5 h. Phage that bound tothe target were captured for 15 min with 200 μl Dynabeads® MyOneStreptavidin that had been previously blocked with Blocking buffer (PBS,0.5% (w/v) bovine serum albumin). Bound phage were eluted with 0.1 M HCland neutralized immediately with 1 M Tris base. Eluted phage wereamplified following the standard protocol as described previously(Sidhu, S. S. et al. Methods Enzymol. 328: 333-363 (2000)). The secondround was carried out the same as the first round using 10 μg ofbiotinylated BACE1 incubated with 400 μl of amplified phage. For allsubsequent rounds, 2 μg biotinylated BACE1 was incubated with 400 μl ofamplified phage. Phage that bound to biotinylated BACE1 were capturedfor 15 min using Maxisorp Immunoplates (NUNC) that had been previouslycoated with Neutravidin or Streptavidin (alternatively between rounds)and blocked with Blocking buffer.

After five rounds of selection, phage were produced from individualclones grown in a 96-well format and the culture supernatants werediluted threefold in phosphate-buffered saline (PBS), 0.5% (w/v) bovineserum albumin (BSA) (Sigma-Aldrich, St Louis, Mo.), 0.1% (v/v) Tween 20(Sigma-Aldrich) (PBT buffer) for use in a phage spot ELISA. The dilutedphage supernatants were incubated for 1 h with biotinylated BACE1 thatwas immobilized on Neutravidin-coated 384-well Maxisorp Immunoplates(NUNC). The plates were washed six times with PBS, 0.05% (v/v) Tween 20(PT buffer) and incubated 30 min with horseradish peroxidase/anti-M13antibody conjugate (1:5000 dilution in PBT buffer) (GE Healthcare). Theplates were washed six times with PT buffer and twice with PBS,developed for 15 min with 3,3′,5,5′-tetramethylbenzidine/H₂O₂ peroxidasesubstrate (Kirkegaard-Perry Laboratories), quenched with 1.0 M H₃PO₄ andthe absorbance was read spectrophotometrically at 450 nm.

Selection of Anti-BACE1 Inhibitory Clones

Panning of the YSGX library resulted in the identification of 18 uniqueclones which bound BACE1. See FIG. 3. The Fab proteins corresponding tothese clones were purified as follows. A stop codon was introducedbetween the heavy chain and gene 3 on the phagemid encoding the Fab. Theresulting phagemid was transformed into E. coli strain 34B8. A singlecolony was grown overnight at 37° C. in 30 ml LB medium supplementedwith 50 μg/ml of carbenicilin. The overnight culture (5 ml) wasinoculated into 500 ml of complete C.R.A.P. medium supplemented withcarbenicilin (50 μg/ml) and grown at 30° C. for 24 h. Fab proteins werepurified using protein A agarose beads by standard methods.

The purified Fabs were screened for inhibitory activity against BACE1using an HTRF enzyme activity assay as described above. Fabs 2, 5, 8,12, 14 and 19 were identified as inhibitors of BACE1 and Fab 23identified as an activator. See FIG. 5.

Fabs 2, 5, 8, 12, 14 and 19 were further characterized to determinetheir binding epitope. The panel of purified Fabs for all 6 antibodieswas used to compete with the individual Fab-displaying phage bound toplate-captured BACE1 in a phage competition ELISA as described below.

Single colonies (in XL1 blue cells) of the selected clones were pickedup and grown in 1 ml 2YT broth supplemented with 50 μg/ml carbenicillin,10 μg/ml tetracycline and M13KO7 at 37° C. for 2 h. Kanamycin (25 μg/ml)was added to the culture, which continued to grow for 6 h. The culturewas transferred to 30 ml 2YT broth supplemented with 50 μg/mlcarbenicillin and 25 μg/ml kanamycin and grown at 37° C. overnight.Phage were harvested and purified as previously described (Sidhu, S. S.et al. Methods Enzymol. 328: 333-363 (2000)). The purifiedFab-displaying phage were serially diluted in PBT buffer and tested forbinding to BACE1 immobilized on a plate. A fixed phage concentrationthat gives 80% of saturation signals was selected for the subsequentcompetition ELISA. The competition was conducted by incubating thefixed, sub-saturating Fab-displaying phage with serial dilutions ofBACE1 for 1 h and then transferred to the BACE1-immobulized plate for 15min to capture unbound phage. The plate was then washed for 8 times andbound phage were detected by anti-M13-HRP.

Purified Fab 5 was competitive with the binding of BACE1 tophage-displayed Fabs 8 and 12, but not to Fabs 2, 14 and 19. Consistentwith this data, purified Fab 8 was competitive with phage-displayed Fabs5 and 12. Taken together, these data indicate that Fabs 5, 8 and 12 bindto the same or overlapping epitopes on BACE1. Fabs 14 and 19 were alsocompetitive with each other based on the fact that either of thesepurified Fabs were competitive with either Fab 14- and 19-displayingphage. This suggested that these two antibodies bind to the same oroverlapping epitope, which differs from the one(s) for Fabs 5, 8 and 12.In the phage ELISA assay, Fab 2-displaying phage could not be competedoff by any of the purified Fab proteins, including Fab 2 itself,suggesting that the binding between Fab 2 and BACE1 was non-specific.Therefore, Fab 2 was excluded as a candidate for affinity maturation.

Affinity Maturation of Anti-BACE1 Inhibitory Clones

To improve the binding affinity of the parent inhibitory antibodiesobtained by the initial panning process, new phage libraries weredesigned that randomized all three CDR-LC of Fabs 5, 8, 12, 14 and 19.These five antibodies were divided into two subgroups based on theirdifferent epitopes—Fabs 5, 8 and 12 as group 1 and Fabs 14 and 19 asgroup 2. Single stranded DNA (ssDNA) for individual clones was purifiedas templates for library construction. The ssDNA templates of group 1were pooled together for affinity maturation library 1 (designed asLC-lib1), and group 2 pooled for library 2 (LC-lib2). The chemicaldiversity was restricted within the randomized CDRs based on thefunctional capacity of the natural amino acids for molecularrecognition. See Birtalan, S. et al. Mol Biosyst. 6:1186-1194 (2010).Minimalist diversity (Tyr and Ser binary codon), semi-minimalistdiversity (Tyr, Ser, Gly and Trp ternary codon) and additional diversitywith 10 amino acids involved were mixed in order to achieve highaffinity. Two affinity maturation libraries, LC-lib1 and LC-lib2, wereconstructed using the same set of oligonucleotide pools to randomize allthree CDR-LC simultaneously as described above. For affinity maturation,all three CDR-LC (Complementarity Determining Region—Light Chain) wererandomized with fixed CDR-HC (Complementarity Determining Region—HeavyChain) for selected clones derived from the primary sorting.

Screening of the libraries for affinity maturation of initially obtainedantibodies was carried out similarly as described above. The librarieswere sorted with biotinylated BACE1 in solution for 3 rounds, whichresulted in greater than 100-fold enrichment in binding. For round 1, 2μg of biotin-BACE1 was incubated with the phage-displayed Fab library.For rounds 2 and 3, 20 nM and 5 nM biotinylated BACE1 was incubated withamplified phage, respectively. Clones (96) from each of the twolibraries were screened in a one-point competition ELISA, where 20 nM ofBACE1 was used in solution to compete the phage particle from binding toplate-immobilized BACE1 as described below.

A plate immobilized with BACE1 was prepared by capturing 2 μg/mlbiotinylated BACE1 for 15 min using a 384-well Maxisorp Immunoplate thatwas previously coated with 2 μg/ml of Neutravidin at 4° C. for overnightand blocked with Blocking Buffer. The culture supernatants fromindividual clones grown in a 96-well format were diluted 20-fold in PBTbuffer and was incubated with or without 20 nM BACE1 at room temperaturefor 1 h. The mixture was transferred to the plate with immobilized BACE1and incubated for 15 min. The plate was washed six times by PT bufferand the bound phage was detected by anti-M13-HRP as described above. Theratio between the ELISA signal from the well in the absence and thepresence of BACE1 in solution indicates the affinity of the clone, wherehigher ratios indicate the higher affinities.

Five clones from LC_lib1 had a ratio of >4 between the ELISA signal fromthe well in the absence of BACE1 to the ELISA signal from the well inthe presence of BACE1 and one clone from LC_lib2 with ratio >3. Twoclones, designated as LC4 and 11, were derived from Fab5; three clones,LC6, LC9 and LC10, from Fab 12, and LC40 from Fab14 (FIG. 6).

To estimate the affinity of these 6 clones, phage competition ELISAswere performed, as described above, and IC₅₀ values were determined(FIG. 7). The IC₅₀ values were determined by fitting the data to afour-parameter logistic equation developed by Marquardt (Marquardt, D.W. SIAM J. Appl. Math. 11: 431-441 (1963)) using Kaleidagraph (SynergySoftware) and are shown below in Table 2.

TABLE 2 Fold affinity FabID IC50 nM improvement Fab5* 20.7 ± 9.5  1 LC40.46 ± 0.07 45 LC11 0.88 ± 0.14 24 Fab12* 45.3 ± 31   1 LC9 0.12 ± 0.02378 LC10 0.12 ± 0.01 378 LC6 0.12 ± 0.02 156 Fab14* 14.3 ± 2.5  1 LC404.7 ± 0.7 3 *parent

All LC clones indeed showed improved affinity compared to theirrespective parents. Notably, the introduction of two Trp residues intoCDR-L2, improved the affinity of Fab 12 derivatives over 100-fold fromthe parent.

Fab proteins of 6 clones were purified and subjected for HTRF enzymeactivity assay, as described above. OM99-2 (CalBiochem®, catalog#496000), a peptide inhibitor of BACE1, was used as a control. For theantibodies with Fab 5 as parent, Fab LC 4 showed significant improvedinhibition, whereas LC11 lost inhibition activity. LC 40, derivativefrom Fab 14, also lost its inhibition activity. The affinity improvedderivatives of Fab 12, Fabs LC 6, LC 9 and LC10, generally showedapproximately 20-fold improvement in their inhibition activity (FIG. 8).Based on this assay, Fab LC 6 was the best inhibitor and showed almost100% inhibition of the enzyme activity, whereas other Fabs were partialinhibitors, having an extent of inhibition of approximately 60-70% (FIG.8). The IC₅₀ values for the various Fabs tested are shown below in Table3. The IC₅₀ OM99-2 was 11 nM in this assay.

TABLE 3 Fab ID IC50 (nM) Fab5* 130 LC4 480 LC11 n.d. Fab12* n.d. LC9 140LC10 180 LC6 160 Fab14* 740 LC40 n.d. *parent

The sequence of the light and heavy chain HVR regions of Fab12 is shownin FIGS. 2(A) and 2(B). The light and heavy chain HVR sequence of threeantibodies produced by affinity maturation of Fab 12 are also shown inFIGS. 2(A) and 2(B). Only light chain HVR-L2 displayed variability inthese antibodies: HVR-L2: X₁₅ Ala Ser X₁₆ Leu Tyr Ser (SEQ ID NO: 41),wherein X₁₅ is selected from serine, tryptophan and tyrosine and X₁₆ isselected from serine and tryptophan. Each of the three heavy chain HVRregions were identical in the four antibodies.

Fabs were cloned as IgG antibodies for use in other applications asfollows. The variable domains of light chain and heavy chain of theselected Fabs were cloned into a pRK5-based plasmid with human lightchain or heavy chain (human IgG1) constant domain for transient IgGexpression in 293T cell or Chinese hamster ovary (CHO) cells. IgGproteins were purified using protein A agarose beads by standardmethods.

Example 2 Further Characterization of Anti-BACE1 Antibodies

As described above antibodies were identified in terms of function andepitope binding on BACE1. The parent and affinity matured antibodieswere further characterized using the assays described below.

A. Binding Kinetics

The binding kinetics of YW412.8.31 was assessed. Briefly, bindingaffinities of anti-BACE1 IgGs were measured by surface plasmon resonance(SPR) using a BIAcore™-3000 instrument. YW412.8.31 anti-BACE1 human IgGwas captured by mouse anti-human Fc antibody (GE Healthcare, cat#BR-1008-39) coated on CM5 biosensor chips to achieve approximately 100response units (RU). For kinetics measurements, two-fold serialdilutions (0.98 nM to 125 nM) of human BACE1 ECD or murine BACE1 ECD(amino acids 1-457) was injected in PBT buffer (PBS with 0.05% Tween 20)at 25° C. with a flow rate of 30 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) were calculated using a simple one-to-oneLangmuir binding model (BIAcore™ Evaluation Software version 3.2). Theequilibrium dissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on). The results of YW412.8.31 binding at pH 7.0 are shown inTable 4.

TABLE 4 Binding kinetics values for anti-BACE1 antibodies as measured byBIAcore ™ TYPE OF K_(ON) K_(OFF) K_(D) ANTIBODY BACE1 (M⁻¹S⁻¹) (S⁻¹) (M)YW412.8.31 Human  1.1 × 10⁵  3.1 × 10⁻⁴  2.9 × 10⁻⁹ 1.05 × 10⁵ 1.39 ×10⁻⁴ 1.32 × 10⁻⁹ YW412.8.31 Mouse  1.4 × 10⁵  2.9 × 10⁻⁴  2.1 × 10⁻⁹1.01 × 10⁵ 1.41 × 10⁻⁴  1.4 × 10⁻⁹ YW412.8.31 Guinea pig  1.0 × 10⁵  2.7× 10⁻⁴  2.6 × 10⁻⁹

Binding of YW412.8.31 to BACE1 was confirmed at both pH 7.0 and 5.0.This is important as BACE1 is optimally active at acidic pH, presumablyin endocytic vesicles and/or the trans-Golgi network.

B. In Vitro Inhibition Assays

Additionally, the ability of antibodies to modulate BACE1 proteolyticactivity on certain BACE substrates was assessed in vitro using twoactivity assays: the HTRF assay and a microfluidic capillaryelectrophoretic (MCE) assay with the human recombinant extracellulardomain of BACE1.

The affinity matured YW412.8.31 anti-BACE1 antibody was tested in a HTRFassay as described in Example 1. A synthetic peptide inhibitor of BACE1,OM99-2 (CalBiochem®, Catalog #496000), a small molecule inhibitor ofBACE1 (β-Secretase inhibitor IV, CalBiochem®, Catalog #5657688) and anIgG antibody which does not bind to BACE1 were used as controls. SeeFIG. 9 (Panel A) (long peptide). Additionally, reactions using a shortFRET peptide (Rh-EVNLDAEFK-quencher (SEQ ID NO:54), Invitrogen) werealso performed identically to the HTRF reactions. The resultingfluorogenic products from the control reactions were measured as above,but at an excitation wavelength of 545 nm and an emission wavelength of585 nm. Obtained data were analyzed using GraphPad Prism 5™ (LaJolla,Calif.). See FIG. 9 (Panel A) (short peptide).

The MCE assay reactions were carried out in a final volume of 20 μL perwell in a 384-well microplate. A standard enzymatic reaction, initiatedby the addition of 10 μL 2× substrate to 5 μL of 4× enzyme and 5 mL of4× compound, containing 12 nM human BACE1 extracellular domain, 1 mMamyloid precursor protein beta secretase active site peptide(FAM-KTEEISEVNLDAEFRWKK-CONH₂ (SEQ ID NO:55)), 50 mM NaOAc pH 4.4 and0.1% CHAPS. The same reaction conditions were used for the extracellulardomain of human BACE2 enzyme (5 nM) and the extracellular domain ofCathepsin D (6 nM, Calbiochem®). After incubation for 60 minutes atambient temperature, the product and substrate in each reaction wereseparated using a 12-sipper microfluidic chip analyzed on an LC3000®(both, Caliper Life Sciences). The separation of product and substratewas optimized by choosing voltages and pressure using the manufacturer'soptimization software. The separate buffer contained 100 mM HEPES pH7.2, 0.015% Brij-35, 0.1% coating reagent #3, 10 mM EDTA and 5% DMSO.The separation conditions used a downstream voltage of −500V, anupstream voltage of −2250V and a screening pressure of −1.2 psi. Theproduct and substrate fluorescence was excited at a wavelength of 488 nmand detected at a wavelength of 530 nm. Substrate conversion wascalculated from the electropherogram using HTS Well Analyzer software(Caliper Life Sciences).

The results from the HTRF and MCE assays, using the YW412.8.31 antibody,are shown in FIG. 9. The observed IC₅₀ of this antibody in the longpeptide assay was 1.7 nM, with a maximal inhibition reaching 77%.Additionally, the YW412.8.31 antibody had an IC₅₀ in the short peptideassay of 17 nM. Further, the YW412.8.31 anti-BACE1 antibody inhibitedBACE1 activity with an IC₅₀ of 80 pM in the microfluidic capillaryelectrophoresis assay, and did not inhibit human BACE2 or cathepsin D, alysosomal aspartyl protease. SPR analysis of the YW412.8.31 antibodyalso confirmed that the antibody does not bind BACE2, the most highlyrelated protease to BACE1. These data together indicate that theYW412.8.31 antibody is a potent and selective BACE1 antagonist. Furthercharacterization of this antibody was performed to better understand itsfunction.

C. Cell-Based Inhibition Assays

To determine whether the observed in vitro inhibitory action of theanti-BACE1 antibodies on APP processing was also present in a cellularcontext, in vivo studies were performed. The ability of the antibodiesto inhibit Aβ₁₋₄₀ production in 293-HEK cells stably expressingwild-type human amyloid precursor protein was assessed as follows.293-APP^(WT) cells were seeded overnight at a density of 3×10⁴cells/well in a 96-well plate. 50 μl of fresh media (DMEM+10% FBS)containing an anti-BACE1 antibody or a control IgG1 antibody wasincubated with the cells for 24 hours at 37° C. A tricyclic smallmolecule BACE1 inhibitor (BACE1 SMI) was also used as a control((Compound 8e—Charrier, N. et al. J. Med. Chem. 51:3313-3317 (2008)).The cellular media was harvested and assayed for the presence of Aβ₁₋₄₀using a Aβ₁₋₄₀ HTRF® assay (CisBio) according to the manufacturer'sinstructions. Aβ₁₋₄₀ values were normalized for cell viability, asdetermined using the CellTiter-Glo Luminescent Cell Viability Assay(Promega). Experiments were performed at least three times, and eachpoint in each experiment was repeated in duplicate. Data was plottedusing a four-parameter non-linear regression curve-fitting program(Kaleidagraph, Synergy Software).

Similar studies were also performed in dorsal root ganglia, corticalneurons and hippocampal neurons isolated from mice. Briefly, dissociatedneuronal cultures were prepared from E13.5 dorsal root ganglia (DRG),E16.5 cortical neurons and E16.5 hippocampal neurons. Neurons were grownfor five days in vitro. Fresh media containing YW412.8.31 anti-BACEantibody or control IgG1 was incubated with the neurons for 24 hours.Media was harvested and assayed for the presence of Aβ₄₀ using the MSD®Rodent/Human (4G8) Aβ40 Ultrasensitive kit according to theManufacturer's instructions. Aβ₄₀ values were normalized for cellviability, as determined using the CellTiter-Glo Luminescent CellViability Assay (Promega). The experiment was performed at least threetimes, and each point was repeated in duplicate. Data was plotted usinga four-parameter non-linear regression curve-fitting program(Kaleidagraph, Synergy Software).

All anti-BACE1 antibodies tested (LC6, LC9, YW412.8, YW412.8.30,YW412.8.31 and YW412.8.51) inhibited Aβ₁₋₄₀ production in APP-expressing293 cells as compared to a non-BACE1 IgG antibody inhibitor (Xolair®).See FIG. 10.

As shown in FIG. 11, the YW412.8.31 anti-BACE1 antibody inhibited Aβ₁₋₄₀production in APP-expressing 293 cells to a similar extent as the BACE1SMI control, with an IC₅₀ of 17 nM and a maximum reduction of ˜90%. Asimilar result was obtained in DRG neurons, with about a 50% reductionin Aβ₄₀ production at the highest concentrations of YW412.8.31, and anIC₅₀ of 8.4 nM. The YW412.8.31 anti-BACE1 antibody also inhibited Aβ₄₀production in cortical and hippocampal neurons with an IC₅₀ of 2.3-2.6nM. These findings indicate that the anti-BACE1 antibodies functionedsimilarly on cells as previously observed in vitro. Furthermore, theYW412.8.31 antibody appears to show the best potency in neurons of theCNS.

D. Intracellular Localization of Anti-BACE1 Antibody

BACE1 is known to be expressed intracellularly, particularly in theGolgi. To ascertain whether or not YW412.8.31 interacts with BACE1 in anintracellular environment, internalization studies were performed. Oneset of neuronal cultures was prepared from E13.5 dorsal root ganglia(DRG) explants, and a second set of neuronal cultures was prepared fromE16.5 dissociated cortical neurons from BACE1+/+ or BACE1−/− mice andcultured for 24 or 72 hours, respectively, at 37° C. Media containing0.5 μM YW412.8.31 anti-BACE1 antibody was added to the cultures for timeperiods varying from 30 minutes to 2 hours, and incubated at either 4°C. or 37° C. Unbound antibody was washed out thoroughly with PBS aftertreatment. Cultures were fixed with 4% paraformaldehyde for 20 minutesat room temperature, and selected samples were also permeabilized with0.1% Triton X-100. Bound antibody was detected using a secondary Alexa568-conjugated anti-Human IgG1 antibody (Molecular Probes) according tothe manufacturer's directions.

The majority of antibody signal was found to be internalized in the hightemperature samples. As can be seen FIG. 12(B), BACE1 can be detectedintracellularly in DRG axons at 37° C. when the cells are permeabilizedto allow for detection of YW412.8.31 anti-BACE1 antibody with thesecondary antibody. Conversely, when DRGs are cold incubated at 4° C. toprevent internalization, or when the cells are not permeabilized toallow for intracellular detection of YW412.8.31, very little BACE1 isdetected at the cell surface. Internalization of the antibody intoneurons was dependent on BACE1 binding, because it was detectable onlyin cortical neurons from BACE1+/+ animals, but not neurons fromBACE1−/−animals (compare center and right panels of FIG. 12(C)).

Additionally, mouse cortical neurons were cultured in the presence ofYW412.8.31 anti-BACE1 antibody or a control IgG for 10 minutes or 3hours, after which the antibody was detected by immunostaining Neuronalcultures were prepared from E15.5 dissociated cortical neurons, andcultured for 14 DIV. Media containing 1 μM YW412.8.31 was added tocultures for 10 minutes to 3 hours, and incubated at 37° C. Unboundantibody was washed out thoroughly with HBSS after treatment. Cultureswere fixed with 2% paraformaldehyde for 10 minutes at RT, and theneither permeabilized with 0.1% Triton X-100, or not. Bound antibody wasdetected using an Alexa 568-conjugated anti-Human IgG1 secondaryantibody (Molecular Probes). YW412.8.31 localization was analyzed innon-permeabilized cells, to see how much was bound on the surface ofcells, as well as in permeabilized cells to see how much antibody wasinternalized. The majority of antibody signal detected was localizedintracellularly, with little antibody staining observed on the cellsurface (FIG. 12(A)). Internalization was evident following only 10minutes of YW412.8.31 treatment, suggesting that the antibody isactively taken up by early endosomes. Much of the YW412.8.31 signal waspunctate indicating it was likely contained within vesicles.

To better identify the subcellular compartments to which YW412.8.31 waslocalized, we co-stained with markers of different vesicularcompartments: early endosomes (transferrin receptor, TfR), trans-golginetwork (TGN) (VAMP4), and lysosome (LAMP1). Cells were co-stained withanti-TfR (Novus, Cat#NB100-64979), anti-VAMP4 (Novus, Cat#NB300-533) oranti-LAMP1 (BD Pharmingen, Cat#553792). YW412.8.31 immunoreactivityco-localized with markers for early endosomes and TGN, but not lysosomes(FIG. 12(A)). This pattern is consistent with antibody localizing tocompartments where BACE1 is active.

Example 3 Anti-BACE1 Antibody Binding Site Characterization

Further studies were performed to identify the binding site of certainanti-BACE1 antibodies to human BACE1. In one set of experiments, thebinding of the antibodies to BACE1 (hBACE1) was assessed in the presenceor absence of known active site or exosite BACE1 binding peptides todetermine which antibodies demonstrated competitive binding. In a secondset of experiments, an anti-BACE1 Fab was co-crystallized with the humanBACE1 extracellular domain to determine the three-dimensional bindingsite.

A. Competitive Binding

As an indirect method of determining the binding site on BACE1 of theanti-BACE1 antibodies of the invention, a competitive ELISA wasperformed. Briefly, antibody YW412.8 IgG (1 μg/ml) was coated onto NUNC96 well Maxisorp immunoplates overnight at 4° C. and blocked at roomtemperature for 1 hour with blocking buffer PBST (PBS and 1% BSA and0.05% Tween 20). Serial dilutions of anti-BACE1 antibody YW412.8 or anhBACE1 binding peptide were incubated with a predetermined amount ofbiotinylated hBACE1 and incubated at room temperature for 60 minutes.The serial dilutions were then added to a YW412.8-coated plate andincubated at room temperature for 30 minutes. Subsequently, the plateswere washed with wash buffer (PBS with 0.05% T-20) and developed by theaddition of streptavidin labeled with horseradish peroxidase (HRP) for30 minutes at room temperature. The plates were then washed anddeveloped with tetramethylbenzidine (TMB) substrate. HRP-conjugatedstreptavidin binding to captured biotinylated hBACE1 was measured at awavelength of 630 nm using standard techniques.

To determine the optimal concentration of biotinylated target proteinused for the above competition ELISA assay, NUNC 96 well Maxisorpimmunoplates were coated and blocked as described above. Serialdilutions of biotinylated target were incubated with antibody-coatedplates for 30 min at room temperature. The plates were then washed withPBST, followed by incubation with horseradish peroxidase conjugatedstrepavidin for 30 minutes at room temperature. Detection of bindingsignal was as described as above. Data was plotted using afour-parameter non-linear regression curve-fitting program(Kaleidagraph, Synergy Software). The sub-saturating concentration ofbiotinylated hBACE1 was determined from the curve fitting and applied tothe competition ELISA from above.

As expected, YW412.8 competed with itself for binding to hBACE1 (FIG.13). No competition was observed between YW412.8 and an active siteinhibitor peptide OM99-2 (CalBiochem®, catalog #496000). Competition wasobserved between the LC6 and YW412.8 anti-BACE1 antibodies and a knownexosite binding peptide, BMS1 (Peptide 1 from Kornacker et al.,Biochemistry 44:11567-11572 (2005)). Combined, these results suggestthat YW412.8 binds at a BACE1 exosite different from the BACE1 activesite for APP cleavage. The shape of the curves in FIG. 13 suggests thatYW412.8, LC6 and BMS1 may have overlapping binding sites on BACE1.

B. Crystal Structure

To better understand the interaction of the YW412.8 antibody with BACE1,the YW412.8.31 Fab was co-crystallized with the extracellular domain ofhuman recombinant BACE1 extracellular domain.

Protein Expression and Purification

Protein expression and purification of BACE1 (amino acids 57-453 of SEQID NO:49) DNA with C-terminal His6 tag (SEQ ID NO: 210) was synthesizedby Blue Heron, cloned into pET29a(+) vector (Novagen), and transformedinto BL21(DE3) cells (Invitrogen). Expression was performed at 37° C.for 4 hours with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG)induction. Cells were lysed with microfluidizer and inclusion bodies(containing BACE1) were isolated and washed two times with TE (10 mMTris pH8.0 and 1 mM ethylenediaminetetraacetic acid (EDTA)) buffer.Protein solubilization was performed using 7.5 M urea, 100 mM AMPSO pH10.8 and 100 mM β-mercaptoethanol (BME) at room temperature for 2 hoursbefore centrifugation at 12,000 rpm for 30 min. The supernatant was thendiluted with 7.5 M urea, 100 mM AMPSO pH 10.8 to achieve an OD₂₈₀ ofabout 1.5-2.0. Protein refolding was performed by first diluting thesolubilized BACE1 1:20 in cold water, then gently stirring the sample at4° C. for 3 weeks to allow refolding to take place. Purification ofrefolded BACE1 involved 3 column chromatography steps. First, BACE1 wasloaded onto a 50 ml Q sepharose Fast Flow (GE Healthcare) columnpre-equilibrated with 20 mM Tris pH 8.0 and 0.4 M urea, and was elutedwith a salt gradient of 0-0.5 M NaCl. Peak fractions were pooled,diluted 5 fold with 20 mM Tris pH 8.0 buffer, and loaded onto aSourceTM15Q column (GE Healthcare). A gradient of 0-0.3 M NaCl was usedto elute BACE1. Fractions containing BACE1 protein were pooled,concentrated and purified further on a Superdex™ S75 column (GEHealthcare) in 25 mM Hepes pH 7.5, 150 mM NaCl.

The YW412.8.31 Fab was expressed in E. coli and the cell paste wasthawed in PBS, 25 mM EDTA and 1 mM PMSF. The mixture was homogenized,passed twice through a microfluidizer, and centrifuged at 12,000 rpm for60 min. The supernatant was then loaded onto a Protein G column at 5ml/min. The column was washed with PBS to base line and the protein waseluted with 0.58% acetic acid. Fractions containing the YW412.8.31 Fabwere pooled and loaded onto a SP-sepharose column equilibrated with 20mM MES, pH 5.5, and the Fab was eluted with a salt gradient of 0 to 0.25M NaCl. The Fab was further purified on a Superdex™ S75 column in 25 mMHepes pH 7.5 and 150 mM NaCl.

Crystallization

Purified BACE1 protein (amino acids 57 to 453 of SEQ ID NO:49) was mixedwith purified YW412.8.31 Fab at a 1:1.5 molar ratio (excess of Fab). Thecomplex was incubated for 1 hour on ice and purified on a 5200 26/60 gelfiltration column (GE Healthcare) to separate it from the excess Fab.The complex was then concentrated to 15 mg/ml. Crystallization was doneby the sitting drop vapor diffusion method with 1 μl of the BACE1/Fabcomplex solution mixed with 1 μl of well solution containing 20% PEG4000, 0.1M Tris pH 8.5 and 0.2M sodium acetate. The crystallizationdrops were then incubated at 19° C. Crystals appeared after 4 days andcontinued to grow for 2 more days. The crystals were then harvested andflash frozen in a cryo-protective solution containing mother liquor and20% glycerol.

Data Collection and Structure Determination

The diffraction data were collected using a monochromatic X-ray beam(12658.4 eV) at the Stanford Synchrotron Radiation Facility (SSRL) beamline 7-1. The X-ray detection device was an ADSC quantum-315 CCDdetector placed 430 mm away from the crystal. Rotation method wasapplied to a single crystal for collection of the complete data set,with 0.5° oscillation per frame and total wedge size of 180°. The datawas then indexed, integrated, and scaled using program HKL2000® (HLKResearch, Inc.).

The structure was solved using the molecular replacement (MR) methodwith the program Phaser (Read, R. J., Acta Cryst. D57:1373-1382 (2000)).Matthews' coefficient calculation results indicated each asymmetric unitwas composed of one BACE1/Fab complex and 48% solvent. Therefore the MRcalculation was directed to search for one set of three subunitsincluding the N- and C-domains of the Fab, and the BACE1 extracellulardomain. The N- and C-terminal Fab domains were searched separately toallow flexible elbow angle. The search models of Fab subunits werederived from the crystal structure of HGFA/Fab complex (PDB code: 2R0L,Wu, Y. et al. Proc. Natl. Acad. Sci. USA 104:19784-19789 (2007)). Thesearch model of BACE1 is from the published BACE1 structure PDB code:1FKN (Hong, L. et al. Science 290:150-153 (2000)). Significantconformational changes take place at the BACE1/Fab interface. Manualrebuilding was done with the program COOT (CrystallographicObject-Orientation Toolkit) (Emsley & Cowtan, Acta. Cryst. D60:2126-2132(2004)). Structure refinement was carried out iteratively with theprogram REFMAC5 (Murshudov, G. N., et al., Acta Cryst. D53:240-255(1997)) and PHENIX (Python-based Hierarchical Environment for IntegratedXtallography) (Adams, P. D. et al. Acta. Cryst. D66:213-221 (2010))using the maximum likelihood target functions, to achieve a final Rfactor of 0.221 and an R_(free) of 0.274. Structure refinementstatistics are shown in Table 5.

TABLE 5 Crystallography Data Statistics Data collection Space group P2₁Unit cell a = 46.1Å, b = 75.5 Å, c = 112.0 Å, a = 90° b = 99.8° g = 90°Resolution 30-2.8 Å Total number of 64939 reflections Completeness 97.9%(84.4%)² Redundancy 3.5 (2.5) I/σ 10.8 (2.0) Rsym¹ 0.112 (0.366)Refinement Resolution range 30-2.8 Å Rcryst³/Rfree⁴ 0.221/0.274 Free Rtest set size 5% of observed reflections Non-hydrogen atoms 6324 Watermolecules 94 Average B, Overall 37.3 Protein 37.6 Water 29.7 r.m.s.dbond lengths 0.003 Å r.m.s.d..angles 0.705° ¹Rsym = Σ|I_(hi) −I_(h)|/ΣI_(hi), where I_(hi) is the scaled intensity of the ithsymmetry-related observation of reflection h and I_(h) is the meanvalue. ²Values in parentheses are of the highest resolution shell whichis (1.97-1.90 Å). ³Rcryst = Σ_(h)|F_(oh) − F_(ch)|/Σ_(h)F_(oh), whereF_(oh) and F_(ch) are the observed and calculated structure factoramplitudes for reflection h. ⁴Value of Rfree is calculated for 5%randomly chosen reflections not included in the refinement.

The crystal diffracted and the structure was refined at 2.8 Åresolution. The overall structure of the BACE1 in the complex largelyresembles its free form (Hong et al., Science 290:150-153 (2000)) whichcan be aligned with 0.63 Å RMSD at the Cα atom positions of 96%(373/385) of the residues. The YW412.8.31 Fab covers a surface area of˜840 Å² on the BACE1 molecular surface and does not bind in the vicinityof the active site. The epitope comprises structural elements denoted byHong et al. (Science 290:150-153 (2000)) as loop C (amino acids 315-318of full-length BACE1), D (amino acids 331-335 of full-length BACE1), andF (amino acids 370-381 of full-length BACE1), which are closely locatedin three-dimensional space. Additionally, the part of BACE1 at and inthe vicinity of the YW412.8.31 binding site adopted conformationalchange, and resulted in a shape complementary score of 0.71, consistentwith strong binding. The antibody induced conformational change isthought to contribute to allosteric inhibition of the secretaseactivity.

The Fab bound to an exosite distal to the active site of BACE1 foramyloid precursor protein, partially overlapping an exosite previouslyidentified as the binding site for a panel of BACE1 binding peptides(Kornacker et al., Biochemistry 44:11567-11572 (2005) (FIG. 14). Boththe heavy and light chains are involved in the interaction (FIG. 15).Unlike the free form, where the BACE1 epitope region is more dynamic asindicated by high temperature factors, the antibody-bound structure isstabilized in a unique confirmation, which deforms the P6 and P7 sites(Turner et al., Biochemistry 44:105-112 (2005)) of the secretase.Adjacent to those sites, amino acids 218-231 (AGFPLNQSEVLASV (SEQ IDNO:126) of SEQ ID NO:49 (residues 157-170 in Lin et al., Proc. Natl.Acad. Sci. USA 97:1456-1460 (2000) (amino acid numbering starts at themature protease domain of BACE1)), which adopt an α-helical structure inthe substrate-bound complex, become a random loop in the antibodycomplex, which adversely impacts APP proteolytic cleavage, perhaps bypreventing APP from reaching into the BACE1 catalytic cleft in acatalytic competent manner. The structural epitope includes the aminoacid residues of BACE1 that contain one or more atoms located within 4angstroms distance from any part of the YW412.8.31 Fab in the crystalstructure. Fab light chain residues belong to chain L, and Fab heavychain residues belong to chain H in Table 6 below. Residue numbering forBACE1 amino acids is based on the full-length sequence of BACE1 (SEQ IDNO:49). Residue numbering for the Fab amino acids is based on the Kabatnumbering scheme (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991).

TABLE 6 Residues located in the YW412.8.31-BACE1 binding interface BACE1Residues Fab Residues 314 SER H 26 GLY 316 GLU H 27 PHE 317 LYS H 28 THR318 PHE H 30 LEU 319 PRO H 31 GLY 327 GLN H 32 TYR 328 LEU H 53 ALA 329VAL H 58 ASP 330 CYS H 94 ARG 331 TRP H 96 PRO 332 GLN H 97 PHE 333 ALAH 98 SER 335 THR H 99 PRO 337 PRO H 100 TRP 340 ILE 375 THR L 49 TYR 378ASP L 53 PHE 380 CYS L 55 TYR 426 PHE L 56 SER L 94 TYR

The detailed atomic interactions are in the form of van der Waalscontacts of polar interactions. Polar interactions include hydrogenbonds and salt bridges. Table 7 below includes a list of the pairwisepolar interactions between BACE1 and the YW412.8.31 Fab. Fab light chainresidues belong to chain L, and Fab heavy chain residues belong to chainH. Residue numbering for BACE1 amino acids is based on the full-lengthsequence of BACE1 (SEQ ID NO:49). Residue numbering for the Fab aminoacids is based on the Kabat numbering scheme.

TABLE 7 Pairwise polar interactions between BACE1 and the YW412.8.31 FabBACE1 Residues---Fab Residues 314 SER---H 98 SER 317 LYS---H 58 ASP 327GLN---H 53 ALA 330 CYS---H 31 GLY 331 TRP---H 98 SER 331 TRP---H 32 TYR332 GLN---H 32 TYR 378 ASP---H 32 TYR 316 GLU---L 94 TYR 332 GLN---L 55TYR 335 THR---L 49 TYR

As shown below, the amino acid composition in the YW412.8.31 antibodyBACE1 epitope is poorly conserved among the corresponding regions inBACE2 and Cathepsin D. This amino acid difference in the epitope for theYW412.8.31 antibody is consistent with the observation that the antibodyis highly selective toward BACE1. Numbering is based on the full-lengthsequence of BACE1 (SEQ ID NO:49). Sequences of BACE2 and Cathepsin D arealigned to BACE1 based on their respective crystal structures. Residuesin the YW412.8.31 BACE1 epitope are boxed.

Example 4 In Vivo Characterization—Mice

The effect of YW412.8.31 in vivo was assessed. To establish the maximalAβ₁₋₄₀ reduction that a BACE1-specific inhibitor could achieve, thecontribution of BACE1 to Aβ₁₋₄₀ production in plasma and forebrain ofBACE1−/− mice compared to BACE1+/+ controls was examined. Plasma Aβ₁₋₄₀signal was reduced by 45%, and brain Aβ₁₋₄₀ signal by 80% in BACE1−/−mice (FIG. 16, panel A). These results imply that BACE1 is indeed themajor β-secretase in the forebrain, but that in the periphery, BACE1accounts for only partial Aβ₁₋₄₀ production, with the remainder comingfrom another β-secretase.

With an understanding of the contribution of BACE1 to Aβ production, theability of the anti-BACE1 antibody YW412.8.31 to modulate amyloidogenicprocessing in hAPP transgenic and wild-type mice was assessed.

hAPP Transgenic Mice

Briefly, 5-month old human APP-expressing mice were treated with 30mg/kg or 100 mg/kg YW412.8.31 antibody or vehicle by intra-peritonealinjection every four days for a total of three doses (i.e., at days 1,5, and 9). Animals were euthanized two hours post the final dose. Serum,plasma, and brains were harvested and processed. Plasma, cortex andhippocampus were analyzed for levels of soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ usingan Amyloid beta (Aβ) ELISA test kit per manufacture's instructions (TheGenetics Company). Pharmacokinetic analysis was performed on serum andbrain homogenates.

The results showed that plasma Aβ₁₋₄₀ and Aβ₁₋₄₂ levels were reduced toapproximately 30% of control levels at both the 30 mg/kg and the 100mg/kg YW412.8.31 antibody dose levels (FIG. 17(A), top panels and FIG.18, panel A). However, in contrast to what was observed in wild-typemice, discussed below, levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ in the brain werereduced by only 15-22% at the 100 mg/kg dose level of the YW412.8.31antibody (FIG. 17(A), bottom panels and FIG. 18, panel A). Theconcentration of YW412.8.31 antibody in the brain of treated animalsincreased in a dose-dependent manner, with an observed concentration ofantibody in the brain in 30 mg/kg-treated animals of 4.8±3.6 nM and anobserved concentration of antibody in the brain in 100 mg/kg-treatedanimals of 14.0±9.3 nM, confirming that a higherintraperitoneally-administered dose of antibody indeed translated into ahigher dose of antibody observed in the brain. Plotting of individualpharmacokinetic versus pharmacodynamic readouts suggested that a PK/PDrelationship exists for this antibody in this model (FIG. 17(B)).

Similar experiments were also performed in which YW412.8.31 anti-BACE1antibody was delivered systemically or directly into hAPP transgenicmice brains by continuous ICV infusion. For ICV delivery, antibody wasdelivered continuously for 7 days via an Alzet osmotic minipump (model2001) implanted unilaterally. The amount of YW412.8.31 antibodydelivered was 0.041 mg/day (low dose) or 0.41 mg/day (high dose); 0.33mg/day Control IgG was delivered to the control group. At euthanasia,plasma, cortex, and hippocampus were harvested and analyzed for levelsof soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ by ELISA (The Genetics Company) followingmanufacturer's instructions.

Table 8 below shows the concentrations of YW412.8.31 antibody in thebrain of mice dosed with 30 mg/kg or 100 mg/kg by systemic delivery or0.041 mg/day and 0.41 mg/day by ICV delivery.

TABLE 8 ANTIBODY ANTIBODY CONCEN- CONCEN- TRATION TRATION DOSE IN BRAIN(μG/G) IN BRAIN (NM) Systemic  30 mg/kg 0.7 4.8 Delivery 100 mg/kg 2.114 ICV Low (0.041 mg/day) 13-25 87-167 Delivery High (0.41 mg/day)110-305 733-2003

However, despite high levels of antibody in the brain followinginfusion, Aβ reduction was modest at 15-23% and was similar to thereduction observed with systemic delivery (FIG. 18, panel B). Thisobservation suggests that high dose systemic injection may be able toreduce Aβ levels in hAPP transgenic mice, however the reduction ismodest. The reduced efficacy in the hAPP transgenic mice is believed tobe a consequence of the animal model, since high concentrations in thebrain, equivalent to the concentration in serum following systemicdelivery, did not further reduce Aβ production. Furthermore, thereduction in the brain in the hAPP transgenic mice is modest compared towhat was observed in wild-type mice and described below. Thus, thetransgenic hAPP mice may not be ideal for studying anti-BACE1 effects invivo. Wild-type mice, are a more appropriate model for antibody efficacyfrom a disease viewpoint as well, as the overwhelming majority of theAlzheimer's patient population carries a wild-type APP allele.

Wild-Type Mice

The ability of anti-BACE1 antibodies YW412.8.31 to modulateamyloidogenic processing was also assessed in wild-type mice. Briefly,experiments were performed as described above. A single dose of controlIgG antibody or YW412.8.31 anti-BACE1 antibody (50 mg/kg) was deliveredsystemically by intravenous (IV) injection to wild-type mice. After 24or 48 hours, plasma and brain samples were harvested and Aβ₁₋₄₀ levelswere analyzed. The concentrations of total mouse Aβ₁₋₄₀ in plasma andbrain were determined using a sandwich ELISA following similarprocedures described below for measuring total anti-BACE1 antibodyconcentrations. Briefly, rabbit polyclonal antibody specific for the Cterminus of Aβ₁₋₄₀ (Millipore, Bedford, Mass.) was coated onto plates,and biotinylated anti-mouse Aβ monoclonal antibody M3.2 (Covance,Dedham, Mass.) was used for detection. The assay had lower limit ofquantification values of 1.96 pg/ml in plasma and 39.1 pg/g in brain. Asis shown in FIG. 16, panel B, Plasma Aβ₁₋₄₀ was reduced by 35% andcortical Aβ₁₋₄₀ was reduced by 20%.

Additional experiments with wild-type C57Bl/6J mice were performed inwhich 100 mg/kg of YW412.8.31, or a control IgG, was administeredsystemically. Levels of Aβ₁₋₄₀ in both the plasma and forebrain oftreated animals four hours after a single intraperitoneal (IP) injectionwere determined. Blood was collected from animals by cardiac puncture toisolate plasma. Following PBS perfusion, the brain was harvested andforebrain from one hemibrain was prepared in PK buffer (1% NP-40 in PBS,with Roche complete protease inhibitors) whereas forebrain from theother hemibrain was homogenized in 5M GuHCL, 50 mM Tris pH 8.0, andfurther diluted in Casein Blocking Buffer (0.25% casein/0.05% sodiumazide, 20 μg/ml aprotinin/5 mM EDTA, pH 8.0/10 μg/ml leupeptin in PBS)for Aβ₁₋₄₀ analysis.

As shown in FIG. 22, panel A, the 100 mg/kg dose was able to reduceplasma Aβ₁₋₄₀ by ˜50% of control levels and similar to BACE1 knockoutlevels described previously. However, no change was detected inforebrain Aβ₁₋₄₀ at 4 hours after administration. This early time pointmay be too soon after administration of YW412.8.31 to see an effect inthe brain. A longer time period post administration may be required toobserve reduced Aβ in the brain, especially since reduction of Aβ isobserved in wild-type mice at a lower dose (50 mg/kg) at 24 hours,described above. YW412.8.31 concentrations in serum were very high,1040±140 μg/mL (6.9±0.9 μM) by 4 hours post administration. YW412.8.31concentrations in brain were much lower at 0.7±0.4 μg/g (4.7±2.7 nM),which represented ˜0.07% of concentration in serum, closelyapproximating the predicted 0.1% steady state penetration of antibodiesinto the CNS (Reiber and Felgenhauer, Clin. Chim. Acta. 163:319-328(1987). Importantly, the antibody concentration achieved in brain,4.7±2.7 nM, is near the cellular IC₅₀ that was previously observed (FIG.11). Thus the anti-BACE1 antibody is highly effective in vivo, asdemonstrated by reduction of plasma Aβ₁₋₄₀ down to levels seen in BACE1knockout mice. However, a single systemic dose did not result in brainreduction by 4 hours post administration to mice, mostly likely becausethe time point was too early to observe any effect.

Additional experiments were performed in order to determine the effectof elevated brain antibody levels through repeated dosing. YW412.8.31antibody, or control IgG, was administered at 30 or 100 mg/kg IP every 4days for a total of 3 doses. In this study the levels of Aβ₁₋₄₀ in boththe plasma and forebrain of treated animals 4 hours post last dose weremeasured. Again, ˜50% reduction in plasma Aβ₁₋₄₀ levels followingmulti-dosing at both 30 and 100 mg/kg was observed (FIG. 22, panel B).Remarkably, a 42% reduction in forebrain Aβ₁₋₄₀ at the high dose ofanti-BACE1 was observed, although no reduction was observed at the lowdose. YW412.8.31 antibody concentrations in serum were 480±210 and1500±440 μg/mL, and concentrations in brain were 0.9±0.6 μg/g (5.9±4.3nM) and 3.0±1.6 μg/g (20±10 nM) following administration at 30 and 100mg/kg given every 4 days, respectively. Thus, as predicted, higherantibody levels in brain resulted in robust reductions in Aβ levels.Notably, there was no difference in peripheral Aβ levels at the 30 mg/kgdose compared to the 100 mg/kg dose, suggesting that a maximalperipheral inhibition at 30 mg/kg was achieved and, thus, simplyreducing peripheral Aβ levels is not sufficient to reduce brain levels.

Additionally, PK data was obtained after dosing with YW412.8.31anti-BACE1 antibody in wild-type and BACE1 knock-out mice. See FIG. 19.A single dose of anti-BACE1 (1 or 10 mg/kg) was delivered via IVinjection to BALB/C mice. Serum PK was analyzed out to 21 dayspost-dose.

Total anti-BACE1 antibody concentrations in mouse serum and brainsamples were measured as follows. Antibody concentrations in mouse serumand brain samples were measured using an enzyme-linked immunosorbentassay (ELISA). NUNC 384 well Maxisorp immunoplates (Neptune, N.J.) werecoated with F(ab′)₂ fragment of donkey anti-human IgG, Fc fragmentspecific polyclonal antibody (Jackson ImmunoResearch, West Grove, Pa.)overnight at 4° C. Plates were blocked with phosphate-buffered saline(PBS) containing 0.5% bovine serum albumin (BSA) for 1 hour at roomtemperature the next day. Each antibody (Control IgG and anti-BACE1) wasused as a standard to quantify the respective antibody concentrations.After washing plates with PBS containing 0.05% Tween 20 using amicroplate washer (Bio-Tek Instruments, Inc., Winooski, Vt.), standardsand samples diluted in PBS containing 0.5% BSA, 0.35 M NaCl, 0.25%CHAPS, 5 mM EDTA, 0.05% Tween 20 and 15 ppm Proclin were incubated onplates for 2 hours at room temperature with mild agitation. Boundantibody was detected with horseradish peroxidase conjugated F(ab′)₂goat anti-human IgG, Fc specific polyclonal antibody (JacksonImmunoResearch). Finally, plates were developed using the substrate3,3′,5,5′-tetramethyl benzidine (TMB) (KPL, Inc., Gaithersburg, Md.).Absorbance was measured at a wavelength of 450 nm with a reference of630 nm on a Multiskan Ascent reader (Thermo Scientific, Hudson, N.H.).Concentrations were determined from the standard curve using afour-parameter non-linear regression program. The assay had lower limitof quantitation (LLOQ) values of 3.12 ng/ml in serum and 15.6 ng/g inbrain.

Free YW412.8.31 antibody concentrations in mice were detected followingsimilar procedures described above using BACE1 ECD as coat and ananti-human IgG, Fc specific antibody (Jackson ImmunoResearch) fordetection. The free anti-BACE1 mouse ELISA had LLOQ values of 0.626ng/ml in serum and 3.13 ng/g in brain

Two separate PK assays were used: an assay to detect all YW412.8.31 inserum (total mAb), and an assay to detect only unbound YW412.8.31 inserum (free mAb). Observed PK kinetics were non-linear, and thedifference in total versus free mAb values for samples where YW412.8.31concentration is <10 μg/mL is suggestive of target-mediated clearance.See FIG. 19, panel A. Furthermore, the difference between total mAb andunbound mAb indicates that some of the YW412.8.31 in serum was likelybound to soluble BACE1. Single dose PK analysis in BACE1+/+, BACE1+/−,and BACE1−/− mice confirms the non-linearity observed in the initialstudy, and indicates that the enhanced clearance is indeedtarget-mediated. BACE1−/− mice show linear PK. See FIG. 19 (Panel B).

Example 5 In Vivo Characterization-Monkey

Cynomolgus monkeys were dosed with control IgG or YW412.8.31 anti-BACE1antibody (30 mg/kg) by IV delivery. Plasma and CSF were sampled up to 7days prior to dosing to set mean baseline Aβ₁₋₄₀ levels in each inindividual animal, and then at various times after dosing. Totalanti-BACE1 or control antibody concentrations in monkey serum and CSFsamples were measured using monkey-adsorbed goat anti-human IgGpolyclonal antibody (Bethyl, Montgomery, Tex.) as both coat anddetection (FIG. 20). Free anti-BACE1 antibody concentrations in monkeyswere determined using BACE1 ECD as coat and the monkey-adsorbed goatanti-human IgG antibody (Bethyl) for detection. Both total and freeanti-BACE1 monkey assays had a LLOQ value of 6.25 ng/ml in serum or CSF.PK is as expected for IgG1 dosed in monkey and shows predicted exposure.

Aβ₁₋₄₀ levels in plasma and CSF from Cynomolgus monkeys tested was alsodetermined. Briefly, the concentrations of total cyno Aβ₁₋₄₀ in plasmawere determined using MSD MA6000 Human (6E10) Abeta Kit (Cat#K111BVE-2,Meso Scale Diagnostics) according to the Manufacturer's instructions.The capture antibody, specific for the C terminus of Aβ₁₋₄₀, waspre-coated on the plates, and Sulfo-Tag anti-A13 monoclonal antibody6E10 was used for detection. The assay had lower limit of quantificationvalues of 49.4 pg/ml in plasma. The concentrations of total cyno Aβ₁₋₄₀in CSF were determined using a sandwich ELISA. Rabbit polyclonalantibody specific for the C terminus of Aβ₁₋₄₀ (cat#AB5737, Millipore,Bedford, Mass.) was coated onto plates, and biotinylated anti-Aβmonoclonal antibody 6E10 (Cat#SIG-39340, Covance, Dedham, Mass.) wasused for detection. The assay had a lower limit of quantification valuesof 15.6 pg/ml in CSF.

As shown in FIG. 21 (Panel A), plasma Aβ₁₋₄₀ levels were reduced ˜50% ofbaseline across all individuals. 50% maximal plasma reductions in Aβwere sustained throughout the 7 day observation period. The serumconcentration-time profile for YW412.8.31 anti-BACE1 antibody appearedsimilar to that observed for the control IgG antibody, suggestingkinetics similar to that of a typical IgG1 dosed in the linear range(FIG. 20, panel A). Peak serum antibody concentrations of ˜800 μg/mLwere observed at the time of first sample collection at 15 minutes postadministration and fell to 232 μg/mL by 7 days post-dose. Notably, atall time points measured after dosing, the serum concentrations ofYW412.8.31 exceeded the cellular IC₅₀ (˜2.5 nM, see FIG. 11).

CSF Aβ₁₋₄₀ levels, as shown in FIG. 21 (Panel B), although variable,showed a reduction up to 50% at 1 and 3 days following dosing followedby a trend back toward baseline Aβ at day 7 post dose. The variabilityin baseline plasma and CSF levels is shown in FIG. 21 (Panels C and D).Baseline plasma levels were fairly uniform across animals, whereas CSFAβ₁₋₄₀ levels were highly variable. Thus, all Aβ₁₋₄₀ measurements werenormalized to baseline for each individual monkey.

These data show that a single dose of YW412.8.31 in monkey significantlyreduces plasma and CSF Aβ levels. In the CSF, YW412.8.31 concentrationsof 0.2-0.3 μg/ml were observed over this time period, which translatesto ˜2 nM (FIG. 20, panel B). From this data, it is inferred that thebrain concentrations of YW412.8.31 are in a similar range. Comparing thePK and PD data, these results show that drug exposure in plasma issufficient to maximally inhibit Aβ production over a 7 day window, whiledrug concentrations in CSF near the cellular IC₅₀ and reduce Aβ levelsin brain transiently at the dose level tested (30 mg/kg). In summary,these data provide strong evidence that systemically administeredanti-BACE1 can reduce BACE1 activity in brain, as determined by CSF Aβmeasurements, in a non-human primate.

Example 6 Affinity Maturation of the YW412.8.31 Antibody

The YW412.8.31 antibody was affinity matured guided by the structuredata provided by the previously described crystal structure. Theantibody residues in contact with BACE1 were mutated in order to enhanceaffinity of the YW412.8.31 antibody. Affinity matured clones produced bythis strategy have the nomenclature YW412.8.31xS. YW412.8.31 affinitymatured clones were also produced via soft randomization targeting ofall CDRs, as described previously, and have the nomenclatureYW412.8.31x. Heavy chain variable sequences and light chain variablesequences for clones which bound BACE1 are depicted in FIGS. 23 (A)-(C)and 24 (A)-(C).

Clones which bound BACE1 were tested for BACE1 protease inhibition in acell-based HTRF assay as described previously in Example 2C. Results ofthe assay are depicted in FIGS. 25A and 25B FIG. 25B shows the resultsof Aβ₁₋₄₀ production (pg/ml) from primary cortical neurons treated for24 hours with various affinity matured anti-BACE1 antibodies at theindicated concentrations. Several of the antibodies tested inhibitedBACE1 at a level similar to that observed with YW412.8.31.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. An isolated antibody, or fragment thereof, that binds to BACE1,wherein the antibody reduces or inhibits the activity of the BACE1polypeptide.
 2. The antibody of claim 1, wherein the antibody binds tothe active site of BACE1.
 3. The antibody of claim 1, wherein theantibody binds to an exosite of BACE1.
 4. The antibody of claim 1,comprising at least one hypervariable region (HVR) sequence selectedfrom the group consisting of SEQ ID NOs: 7-19, 22-26, 28-30, 35-47,56-79 and 118-122.
 5. The antibody of claim 1, comprising at least onesequence selected from the group consisting of HVR-H1, HVR-H2 andHVR-H3, wherein HVR-H1 comprises the amino acid sequenceGFX₃₀FX₃₁X₃₂X₃₃X₃₄IH (SEQ ID NO:45), wherein X₃₀=N or T; X₃₁=S, L or Y;X₃₂=G or Y; X₃₃=Y or S; and X₃₄=A, G or S; HVR-H2 comprises the aminoacid sequence X₃₅X₃₆ISPX₃₇X₃₈GX₃₉TX₄₀YADSVKG (SEQ ID NO:46), whereinX₃₅=A or G; X₃₆=W or S; X₃₇=A or Y; X₃₈=G or S; X₃₉=S or Y; and X₄₀=D orS; and HVR-H3 comprises the amino acid sequenceX₄₁PX₄₂X₄₃X₄₄X₄₅X₄₆X₄₇MDY (SEQ ID NO:47), wherein X₄₁=Q or G; X₄₂=T orF; X₄₃=H or S; X₄₄=Y or P; X₄₅=Y or W; X₄₆=Y or V and wherein X₄₇optionally includes the sequence YAKGYKA (SEQ ID NO:48).
 6. The antibodyof claim 1, comprising at least one sequence selected from the groupconsisting of HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises theamino acid sequence GX₇₁X₇₂X₇₃X₇₄X₇₅X₇₆X₇₇IH (SEQ ID NO:120), whereinX₇₁=F or Y; X₇₂=F, N or T; X₇₃=F or Y; X₇₄=L, Q, I, S or Y; X₇₅=G or Y;X₇₆=Y or S; and X₇₇=A, G or S; HVR-H2 comprises the amino acid sequenceX₇₈X₇₉ISPX₈₀X₈₁GX₈₂X₈₃X₈₄YADSVKG (SEQ ID NO:121), wherein X₇₈=A or G;X₇₉=W or S; X₈₀=A, S, Q or Y; X₈₁=G or S; X₈₂=S, K, L or Y; X₈₃=T or Y;and X₈₄=D or S; and HVR-H3 comprises the amino acid sequenceX₈₅PX₈₆X₈₇X₈₈X₈₉X₉₀X₉₁MDY (SEQ ID NO:122), wherein X₈₅=Q or G; X₈₆=T orF; X₈₇=H, Y or S; X₈₈=Y or P; X₈₉=Y or W; X₉₀=Y or V and wherein X₉₁optionally includes the sequence YAKGYKA (SEQ ID NO:48).
 7. The antibodyof claim 1, comprising at least one sequence selected from the groupconsisting of HVR-H1, HVR-H2 and HVR-H3, wherein HVR-H1 comprises theamino acid sequence GX₅₃X₅₄X₅₅X₅₆GYGIH (SEQ ID NO:68), wherein X₅₃=F orY; X₅₄=T or F; X₅₅=F or Y; X₅₆=L, Q or I, HVR-H2 comprises the aminoacid sequence GWISPX₅₇X₅₈GX₅₉X₆₀DYADSVKG (SEQ ID NO:69), wherein X₅₇=A,S or Q; X₅₈=G or S; X₅₉=S, K or L; X₆₀=T or Y, wherein HVR-H3 sequencecomprising the amino acid sequence GPFX₆₁PWVMDY (SEQ ID NO:70), whereinX₆₁=S or Y or an amino acid sequence of SEQ ID NO:79.
 8. The antibody ofclaim 5, comprising an HVR-H1 sequence comprising the amino acidsequence GFTFX₁₃GYX₁₄IH (SEQ ID NO:26), wherein X₁₃=S or L and X₁₄=A orG.
 9. The antibody of claim 6, comprising an HVR-H1 sequence comprisingan amino acid sequence selected from the group consisting of SEQ IDNO:22; SEQ ID NO:23; SEQ ID NO:28 and SEQ ID NOs:71-73.
 10. The antibodyof claim 6, comprising an HVR-H2 sequence comprising an amino acidsequence selected from SEQ ID NO:24, SEQ ID NO:29 and SEQ ID NOs:74-78.11. The antibody of claim 6, comprising an HVR-H3 sequence comprising anamino acid sequence selected from SEQ ID NO:25; SEQ ID NO:30 and SEQ IDNO:79.
 12. The antibody of claim 6, comprising HVR-H1, HVR-H2, andHVR-H3 sequences corresponding to those set forth for clones YW412.8,YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29 and YW412.8.51 in FIG.1(B) or clones in FIGS. 24(A)-(C).
 13. The antibody of claim 6,comprising HVR-H1, HVR-H2, and HVR-H3 sequences corresponding to thoseset forth for clones Fab12, LC6, LC9 and LC10 in FIG. 2(B).
 14. Theantibody of claim 6, comprising an HVR-H1 sequence of SEQ ID NO:22 or23, an HVR-H2 sequence of SEQ ID NO:24 and an HVR-H3 sequence of SEQ IDNO:25.
 15. The antibody of claim 14, wherein the HVR-H1 sequence is SEQID NO:23.
 16. The antibody of claim 6, comprising an HVR-H1 sequence ofSEQ ID NO:28, an HVR-H2 sequence of SEQ ID NO:29, and an HVR-H3 sequenceof SEQ ID NO:30.
 17. The antibody of claim 6, comprising a VH chainhaving an amino acid sequence selected from the group consisting of SEQID NOs: 20, 21, 27 and 80-98.
 18. The antibody of claim 16, wherein theVH chain amino acid sequence is SEQ ID NO:21.
 19. The antibody of claim1, comprising at least one sequence selected from the group of HVR-L1,HVR-L2 and HVR-L3 wherein HVR-L1 comprises the amino acid sequenceRASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:42), wherein X₁₇=S, D or V; X₁₈=S or A;X₁₉=S, T or N; X₂₀=A or S; X₂₁=V or L, HVR-L2 comprises the amino acidsequence X₂₂ASX₂₃LYS (SEQ ID NO:43), wherein X₂₂=S, W, Y or L; X₂₃=F, Sor W, and HVR-L3 comprises the amino acid sequence QQX₂₄X₂₅X₂₆X₂₇X₂₈X₂₉T(SEQ ID NO:44), wherein X₂₄=S, F, G, D or Y; X₂₅=Y, P, S or A; X₂₆=Y, Tor N; X₂₇=T, Y, D or S; X₂₈=P or L; and X₂₉=F, P or T.
 20. The antibodyof claim 1, comprising at least one sequence selected from the group ofHVR-L1, HVR-L2 and HVR-L3 wherein HVR-L1 comprises the amino acidsequence RASQX₁₇VX₁₈X₁₉X₂₀X₂₁A (SEQ ID NO:42), wherein X₁₇=S, D or V;X₁₈=S or A; X₁₉=S, T or N; X₂₀=A or S; X₂₁=V or L, HVR-L2 comprises theamino acid sequence X₆₂ASX₆₃X₆₄YX₆₅ (SEQ ID NO:118), wherein X₆₂=S, W,Y, F or L; X₆₃=F, S, Y or W; X₆₄=L or R; X₆₅=S, P, R, K or W, and HVR-L3comprises the amino acid sequence QQX₆₆X₆₇X₆₈X₆₉X₇₀X₇₁T (SEQ ID NO:119),wherein X₆₆=S, F, G, D or Y; X₆₇=Y, P, S or A; X₆₈=Y, T or N; X₆₉=T, Y,D or S; X₇₀=P, Q, S, K or L; and X₇₁=F, P or T.
 21. The antibody ofclaim 1, comprising at least one sequence selected from the groupconsisting of HVR-L1, HVR-L2 and HVR-L3, wherein HVR-L1 comprises theamino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V;X₂=S or A; X₃=T or N; X₄=S or A; X₅=V or L, HVR-L2 comprises the aminoacid sequence X₄₈ASX₄₉X₅₀YX₅₁ (SEQ ID NO:56), wherein X₄₈=S or F; X₄₉=For Y; X₅₀=L or R; X₅₁=S, P, R, K or W and HVR-L3 comprises the aminoacid sequence QQFPTYX₅₂PT (SEQ ID NO:57), wherein X₅₂=L, Q, S or K. 22.The antibody of claim 19, comprising an HVR-L1 sequence comprising theamino acid sequence RASQX₁VX₂X₃X₄X₅A (SEQ ID NO:17), wherein X₁=D or V;X₂=S or A; X₃=T or N; X₄=S or A; X₅=V or L.
 23. The antibody of claim19, comprising an HVR-L2 sequence comprising the amino acid sequenceX₆ASFLYS (SEQ ID NO:18) or X₁₅ASX₁₆LYS (SEQ ID NO:41), wherein X₆=S orL; X₁₅=S, W or Y; and X₁₆=S or W.
 24. The antibody of claim 19,comprising an HVR-L3 sequence comprising the amino acid sequenceQQX₇X₈X₉X₁₀X₁₁X₁₂T (SEQ ID NO:19), wherein X₇=S, F, G, D or Y; X₈=Y, P,S, or A; X₉=T or N; X₁₀=T, Y, D or S; X₁₁=P or L; X₁₂=P or T.
 25. Theantibody of claim 19, comprising an HVR-L1 sequence comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:7, SEQ IDNO:8 or SEQ ID NO:35.
 26. The antibody of claim 20, comprising an HVR-L2sequence comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NOs:36-39 and SEQ IDNOs:58-64.
 27. The antibody of claim 20, comprising an HVR-L3 sequencecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs:11-16, SEQ ID NO:40 and SEQ ID NOs:65-67.
 28. The antibody ofclaim 20, comprising HVR-L1, HVR-L2 and HVR-L3 sequences correspondingto those set forth for clones YW412.8, YW412.8.31, YW412.8.30,YW412.8.2, YW412.8.29 and YW412.8.51 in FIG. 1(A) and clones in FIGS.23(A)-(C).
 29. The antibody of claim 20, comprising HVR-L1, HVR-L2 andHVR-L3 sequences corresponding to those set forth for clones Fab12, LC6,LC9 and LC10 in FIG. 2(A).
 30. The antibody of claim 20, comprising anHVR-L1 sequence of SEQ ID NO:7 or SEQ ID NO:8; an HVR-L2 sequence of SEQID NO:9 or SEQ ID NO:10; and an HVR-L3 sequence of selected from thegroup consisting of: SEQ ID NOs:11-16.
 31. The antibody of claim 30,wherein the HVR-L1 sequence is SEQ ID NO:7, the HVR-L2 sequence is SEQID NO:9 and the HVR-L3 sequence is SEQ ID NO:12.
 32. The antibody ofclaim 20, comprising an HVR-L1 sequence of SEQ ID NO:35; an HVR-L2sequence selected from the group consisting of SEQ ID NOs:36-39 and anHVR-L3 sequence of SEQ ID NO:40.
 33. The antibody of claim 20,comprising a VL chain sequence having an amino acid sequence selectedfrom the group consisting of: SEQ ID NOs: 1-6, 31-34 and 99-117.
 34. Theantibody of claim 33, wherein the VL chain amino acid sequence is SEQ IDNO:2.
 35. The antibody of claim 15, further comprising an HVR-L1comprising the amino acid sequence of SEQ ID NO:7, an HVR-L2 comprisingthe amino acid sequence of SEQ ID NO:9 and an HVR-L3 comprising theamino acid sequence of SEQ ID NO:12.
 36. The antibody of claim 34,further comprising a VH chain comprising the amino acid sequence of SEQID NO:21.
 37. An isolated antibody, or fragment thereof, which binds toan epitope comprising at least one of the amino acid residues of BACE1selected from the group consisting of: 314 SER; 316 GLU; 317 LYS; 327GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49.38. The antibody of claim 37, wherein the epitope comprises 314 SER; 316GLU; 317 LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASPof SEQ ID NO:49.
 39. An isolated antibody, or fragment thereof, whichbinds to an epitope comprising at least one amino acid region of BACE1selected from the group consisting of: amino acids 315-318 of SEQ IDNO:49; amino acids 331-335 of SEQ ID NO:49; amino acids 370-381 of SEQID NO:49; and any combination thereof.
 40. The antibody of claim 39,wherein the epitope comprises amino acids 315-318, 331-335 and 370-381of SEQ ID NO:49.
 41. An isolated antibody, or fragment thereof, whichbinds to an epitope of BACE1 and results in a conformational change inthe structure of the P6 and P7 sites of BACE1 upon binding.
 42. Anisolated antibody, or fragment thereof, which binds to an epitope ofBACE1 and induces amino acids 218-231 of SEQ ID NO:49 to adopt a randomloop structure upon binding.
 43. The antibody of claim 37, wherein theantibody reduces or inhibits the activity of BACE1.
 44. The antibody ofclaim 4, which is a monoclonal antibody.
 45. The antibody of claim 4,which is a human, humanized, or chimeric antibody.
 46. The antibody ofclaim 4, which is an antibody fragment.
 47. The antibody of claim 4,which is a full length IgG1 antibody.
 48. An isolated nucleic acidencoding the antibody of claim
 4. 49. A host cell comprising the nucleicacid of claim
 48. 50. A method of producing an antibody comprisingculturing the host cell of claim 49 so that the antibody is produced.51. An immunoconjugate comprising the antibody of claim 4 and acytotoxic agent.
 52. A pharmaceutical formulation comprising theantibody of claim 4 and a pharmaceutically acceptable carrier.
 53. Amethod of treating an individual having a neurological disease ordisorder comprising administering to the individual an effective amountof the antibody of claim
 4. 54. A method of reducing amyloid plaques ina patient suffering from, or at risk of contracting, a neurologicaldisease or disorder comprising administering to the individual aneffective amount of the antibody claim
 4. 55. A method of inhibitingamyloid plaque formation in a patient suffering from, or at risk ofdeveloping, a neurological disease or disorder comprising administeringto the individual an effective amount of the antibody of claim
 4. 56.The method of claim 53, wherein the neurological disease or disorder isselected from the group consisting of Alzheimer's disease (AD),traumatic brain injury, stroke, glaucoma, dementia, muscular dystrophy(MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),cystic fibrosis, Angelman's syndrome, Liddle syndrome, Paget's disease,traumatic brain injury, Lewy body disease; postpoliomyelitis syndrome,Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease,multiple system atrophy, striatonigral degeneration, supranuclear palsy,bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome,kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease,fatal familial insomnia, bulbar palsy, motor neuron disease, Canavandisease, Huntington's disease, neuronal ceroid-lipofuscinosis,Alexander's disease, Tourette's syndrome, Menkes kinky hair syndrome,Cockayne syndrome, Halervorden-Spatz syndrome, lafora disease, Rettsyndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, andUnverricht-Lundborg syndrome, Pick's disease, and spinocerebellarataxia.
 57. The method of claim 56, wherein the neurological disease ordisorder is selected from the group consisting of Alzheimer's disease,stroke, traumatic brain injury and glaucoma.
 58. A method of reducingamyloid-β (Aβ) protein in a patient comprising administering to thepatient an effective amount of the antibody of claim
 4. 59. The methodof claim 58, wherein the patient is suffering from, or at risk ofcontracting, a neurological disease or disorder.
 60. The method of claim59, wherein the neurological disease or disorder is selected from thegroup consisting of: Alzheimer's disease, stroke, traumatic brain injuryand glaucoma.
 61. A method of diagnosing a neurological disease ordisorder in patient comprising contacting a biological sample isolatedfrom the patient with an antibody of claim 4 under conditions suitablefor binding of the antibody to a BACE1 polypeptide, and detectingwhether a complex is formed between the antibody and the BACE1polypeptide.
 62. A method of determining whether a patient is eligiblefor therapy with an anti-BACE1 antibody, comprising contacting abiological sample isolated from the patient with an antibody of claim 4under conditions suitable for binding of the antibody to a BACE1polypeptide, and detecting whether a complex is formed between theantibody and the BACE1 polypeptide, wherein the presence of a complexbetween the antibody and BACE1 is indicative of a patient eligible fortherapy with an anti-BACE1 antibody.
 63. The method of claim 61, whereinthe biological sample is selected from the group consisting of serum,plasma, saliva, gastric secretions, mucus, cerebrospinal fluid,lymphatic fluid, neuronal tissue, brain tissue, cardiac tissue orvascular tissue. 64.-69. (canceled)
 70. A BACE1 epitope which isspecifically recognized by a antibody, or fragment thereof, comprisingat least one of the amino acid residues of BACE1 which correspond to theamino acids selected from the group consisting of: 314 SER; 316 GLU; 317LYS; 327 GLN; 330 CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ IDNO:49.
 71. The BACE1 eptiope of claim 70, wherein the eptiope comprisesamino acids with correspond to 314 SER; 316 GLU; 317 LYS; 327 GLN; 330CYS; 331 TRP; 332 GLN; 335 THR; and 378 ASP of SEQ ID NO:49.
 72. A BACE1epitope which is specifically recognized by an antibody, or fragmentthereof, comprising at least one amino acid region of BACE1 selectedfrom the group consisting of: amino acids 315-318 of SEQ ID NO:49; aminoacids 331-335 of SEQ ID NO:40; amino acids 370-381 of SEQ ID NO:49; andany combination thereof.
 73. The BACE1 epitope of claim 72, wherein theepitope comprises amino acids 315-318, 331-335 and 370-381 of SEQ IDNO:49.